Compositions comprising lipase and sulfite

ABSTRACT

The present invention relates to a composition comprising: (a) a source of sulfite; and (b) a lipase variant of a parent lipase, wherein one or more amino acids in the parent lipase within 20 angstrom (Å) of a cysteine bridge are replaced with a more negatively charged amino acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2018/061492 filed May 4, 2018 andpublished on Nov. 8, 2018 as WO 2018/202846, which claims priority orthe benefit under 35 U.S.C. 119 of European application no. 17169748.5filed May 5, 2017, the contents of which are fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions comprising a source ofsulfites and a lipase variant. The invention also relates to lipasevariants and to the use of the composition of the invention forcleaning, e.g., a surface. Further, the invention also relates topolynucleotides, nucleic acid constructs comprising a polynucleotide ofthe invention, expression vectors comprising a polynucleotide of theinvention, host cells comprising a polynucleotide of the invention,methods of producing a lipase variant of the invention and methods forobtaining lipase variants of the invention.

Description of the Related Art

Sulfites are substances that naturally occur in some foods and the humanbody. They are used in food as preservative or enhancer as well as innon-food such as e.g. house hold products, and are thus comprised invarious compositions.

Lipases have been employed in compositions for the removal of lipidstains by hydrolyzing triglycerides to generate fatty acids. Currentdetergent, cleaning and/or fabric care compositions comprise many activeingredients which are interfering with the ability of lipase to removelipid stains. In some compositions sources of sulfites are added asantioxidants towards more labile functional ingredients, such as but notlimited to colors, polymers, perfumes or as traces/impurities insulfonated surfactants. However, sulfites may affect the stability oflipases.

WO 2017/005640 concerns compositions comprising (a) a source of sulfite;and (b) a lipase variant of a parent lipase with improved stabilitytowards sulfite.

A need exists for compositions, including detergent compositions,comprising sulfite and a lipase that are stable and active in thepresence of sulfite.

SUMMARY OF THE INVENTION

The present invention concerns the problem of reduced stability oflipases in compositions comprising a sulfite source.

Sulfite (SO₃ ²⁻) is present in many detergent compositions. Sulfite canbreak di-sulfide bonds (R—S—S—R′) and reduce the stability of lipasescontaining one of more cysteine bridges. The inventors have surprisingfound that loss of stability can be reduced by shielding the cysteinebridges with negatively charged amino acids close to the cysteinebridge. It is believed that this effect is due to the negative charge ofsulfite which is therefore less prone to bind to areas of the lipasebeing negative charged.

Therefore, in the first aspect, the present invention relates tocompositions comprising:

(a) a source of sulfite;

(b) a lipase variant of the parent lipase shown as SEQ ID NO: 2 or of aparent lipase having at least 60% identity to SEQ ID NO: 2;

wherein one or more amino acids in the parent lipase within 20 angstrom(A) of a cysteine bridge are replaced with a more negatively chargedamino acid.

In one embodiment, the parent lipase is the polypeptide of SEQ ID NO: 2;SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12, or a fragment thereof with lipase activity.

In a preferred embodiment, the replaced, preferably substituted, aminoacid is within 15 angstrom, preferably within 10 angstrom, morepreferably within 5 angstrom (i.e., from carboxyl group to sulfur atom)of a cysteine bridge.

In a preferred embodiment, the source of sulfite is selected from:sulfites, such as sodium sulfite, potassium sulfite; calcium sulfite;bisulfite such as potassium bisulfite sodium bisulfite, calciumbisulfite; metabisulfite such as potassium metabisulfite, sodiummetabisulfite, calcium metabisulfite; or any combination thereof.

In a preferred embodiment, the amount of sulfite (as SO₃ ²⁻) ormetabisulfite (as S₂O₅ ²⁻) is from 0.1 wt % to 3 wt %; from 0.2 wt % to2 wt %; from 0.5 wt % to 2 wt %.

In a specific embodiment, the lipase variant comprises one or moresubstitutions corresponding to Q15D, E; G23D, E; T35D, E; T37D, E; N39D,E; A40D,E; P42D,E; N101D,E; S105D, E; G106D, E; F184D, E or T267D, E ofSEQ ID NO: 2.

According to the invention the parent lipase may comprise one or morecysteine bridges.

Said cysteine bridges are preferably located on the surface of theparent lipase.

In a specific embodiment of the invention the cysteine bridge is orcorresponds to one or more of the cysteine bridges in positions:

C22-C268,

C36-C41, and

C104-C107 of SEQ ID NO: 2.

In an embodiment of the invention the lipase variant of the inventionhas improved stability in the presence of a source sulfite as comparedto the parent lipase.

In a preferred embodiment, the composition further comprising asurfactant.

In an aspect, the invention relates to the use of the composition of theinvention for cleaning, e.g., a surface.

The invention also relates to lipase variants of a parent lipase,wherein said variant has lipase activity, has at least 60%, but lessthan 100% sequence identity to SEQ ID NO: 2; or any fragment thereofwith lipase activity, and comprises one or more (e.g. several)substitutions at positions corresponding to residues 15, 23, 35, 37, 39,40, 42, 101, 105, 106, 184, and 267 of SEQ ID NO: 2.

In a specific embodiment, the lipase variant includes one or moresubstitutions corresponding to substitutions selected from the group:Q15D, E; G23D, E; T35D, E; T37D, E; N39D, E; A40D, E; P42D, E; N101D, E;S105D, E; G106D, E; F184D, E and T267D, E of SEQ ID NO: 2.

In an aspect, the invention relates to polynucleotides encoding thevariant of the invention.

In an aspect, the invention relates to nucleic acid constructscomprising a polynucleotide of the invention.

In an aspect, the invention relates to expression vectors comprising apolynucleotide of the invention.

In an aspect, the invention relates to host cells comprising apolynucleotide of the invention, in particular an expression vector ofthe invention.

In an aspect, the invention relates to methods of producing a lipasevariant, comprising: (a) cultivating the host cell of the inventionunder conditions suitable for expression of the variant; and (b)recovering the variant.

In an aspect, the invention relates to methods for obtaining the lipasevariant of the invention, comprising introducing into the parent lipaseshown as SEQ ID NO: 2 or a parent lipase having at least 60% sequenceidentity thereto within 20 angstrom (A) of a cysteine bridge a morenegatively charged amino acid.

In one embodiment, the parent lipase is the polypeptide of SEQ ID NO: 2;SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12, or a fragment thereof with lipase activity.

In a preferred embodiment one or more substitutions corresponding to thefollowing substitutions in SEQ ID NO: 2 are introduced into the parentlipase: Q15D, E; G23D,E; T35D, E; T37D,E; N39D,E; A40D,E; P42D,E;N101D,E; S105D,E; G106D,E; F184D,E; or T267D,E.

DEFINITIONS

Lipase: The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipidesterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to anenzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may havelipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity(EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-esterhydrolase activity (EC3.1.1.50). For purposes of the present invention,lipase activity is determined according to the procedure described inthe Example section: Hydrolytic activity may be determined with a PnPassay using substrates with various chain lengths.

In one aspect, the variants of the present invention have at least 20%,e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100% of the lipase activity of the parent lipase. In one aspect,the parent lipase is the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12, or afragment thereof with lipase activity.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a polypeptide; wherein the fragment has lipase activity. Inone aspect, a fragment contains at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the number of the amino acidspresent in the parent lipase. In one aspect, the parent lipase is thepolypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;or SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect the parent lipase isamino acid 1-269 of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO:8; or SEQ ID NO: 10; or SEQ ID NO: 12.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parentlipase. Such improved properties include, but are not limited to,stability such as e.g. stability in the presence of a source of sulfite,stability in detergent compositions, stability in detergent compositionscomprising a source of sulfite, stability under storage conditions,stability under storage conditions in the presence of a source sulfite,thermostability and thermostability in the presence of a source sulfite.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” or “mature part of apolypeptide” means a polypeptide in its final form following translationand any post-translational modifications, such as N-terminal processing,C-terminal truncation, glycosylation, phosphorylation, etc. In oneaspect, the mature polypeptide is amino acids 1 to 269 of SEQ ID NO: 2;SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12. It is known in the art that a host cell may produce a mixture oftwo of more different mature polypeptides (i.e., with a differentC-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving lipase activity. In one aspect, the mature polypeptide codingsequence is nucleotides 1 to 807 of SEQ ID NO: 1; SEQ ID NO: 3, SEQ IDNO: 5; SEQ ID NO: 7; SEQ ID NO: 9; or SEQ ID NO: 11.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent lipase: The term “parent” or “parent lipase” means alipase to which an alteration is made to produce the lipase variants ofthe present invention. The parent may be a naturally occurring(wild-type) polypeptide or a variant or fragment thereof. Examples ofsuch parent lipases are those with amino acid sequences given in SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; or SEQID NO: 12, or a fragment thereof with lipase activity.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the −nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of apolypeptide coding sequence; wherein the subsequence encodes a fragmentwith lipase activity. In one aspect, a subsequence contains at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95% but lessthan 100% of the number of nucleotides 1 to 807 encoding the parentlipase. In one aspect, the nucleotides encoding the parent lipasecomprises or consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a subsequence thereof encodinga fragment with lipase activity.

Sulfite: The terms “sulfite” means a compound that contain the sulfiteion [SO₃]²⁻. The terms “bisulfite” or “hydrogen sulfite” means acompound that contains the bisulfite ion [HSO₃]⁻. The terms“metabisulfite” or “disulfite” means a compound that contains themetabisulfite ion [S₂O₅]²⁻. For the purpose of this invention use of theterm “source of sulfite” covers any sources of sulfite that provides thecompounds mentioned in the present paragraph. The present inventionincludes sources of sulfite added intentionally.

Variant: The term “variant” means a polypeptide having lipase activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position. The variantsof the present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% of the lipase activity of the polypeptide of theparent lipase. In one aspect, the parent lipase comprises or consists ofSEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO:10; SEQ ID NO: 12, or a fragment thereof with lipase activity.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200ug/mL sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type lipase: The term “wild-type” lipase means a lipase expressedby a naturally occurring microorganism, such as a bacterium, yeast, orfilamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the polypeptide of SEQ ID NO: 2is used to determine the corresponding amino acid residue in anotherlipase. The amino acid sequence of another lipase is aligned with thepolypeptide of SEQ ID NO: 2, and based on the alignment, the amino acidposition number corresponding to any amino acid residue in thepolypeptide of SEQ ID NO: 2 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in another lipasecan be determined by an alignment of multiple polypeptide sequencesusing several computer programs including, but not limited to, MUSCLE(multiple sequence comparison by log-expectation; version 3.5 or later;Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066;Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh,2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods inMolecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), usingtheir respective default parameters.

When the other enzyme has diverged from the polypeptide of SEQ ID NO: 2such that traditional sequence-based comparison fails to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615),other pairwise sequence comparison algorithms can be used. Greatersensitivity in sequence-based searching can be attained using searchprograms that utilize probabilistic representations of polypeptidefamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the polypeptide has one ormore representatives in the protein structure databases. Programs suchas GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin andJones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample, the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly, the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.Multiple variants may also be disclosed as, e.g., “R170Y G195E” (i.e.,without “+” between the alterations).

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants:

“Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and“Tyr167Ala+Arg 170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition comprising: (a) a sourceof sulfite; and (b) a lipase variant of a parent lipase, as well as useof the composition for cleaning, e.g., a surface. The present inventionalso relates to lipase variants, polynucleotides encoding the variants;nucleic acid constructs, vectors, and host cells comprising thepolynucleotides; and methods of producing the variants.

Compositions

The invention provides compositions comprising:

(a) a source of sulfite;

(b) a lipase variant of the parent lipase shown as SEQ ID NO: 2 or of aparent lipase having at least 60% identity to SEQ ID NO: 2;

wherein one or more amino acids in the parent lipase within 20 angstrom(A) of a cysteine bridge are replaced with a more negatively chargedamino acid.

In one embodiment, the parent lipase is the polypeptide of SEQ ID NO: 2;SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12, or a fragment thereof with lipase activity.

In preferred embodiments, the replaced, preferably substituted, aminoacid is within 15 angstrom, preferably within 10 angstrom, morepreferably within 5 angstrom (i.e., from carboxyl group to sulfur atom)of a cysteine bridge.

In an embodiment, the source of sulfite is selected from sulfitesincluding sodium sulfite, potassium sulfite; calcium sulfite; bisulfitesuch as potassium bisulfite sodium bisulfite, calcium bisulfite;metabisulfite such as potassium metabisulfite, sodium metabisulfite,calcium metabisulfite; or any combination thereof.

In an embodiment, the amount of sulfite (as SO₃ ²⁻) or metabisulfite (asS₂O₅ ²⁻) ranges from 0.005 wt % to 5 wt %; such as from 0.01 wt % to 3wt %; such as from 0.1 wt % to 2 wt %; such from 0.2 wt % to 1 wt %;such as from 0.4 wt % to 0.6 wt %; such as from 0.45 wt % to 0.55 wt %;such as 0.005 wt %; such as 0.01 wt %; such as 0.1 wt %; such as 0.25 wt% such as 0.5 wt % or any combination of these ranges or values.

In an embodiment, the composition of the invention comprise a lipasevariant comprises one or more (e.g. several) substitutions at positionscorresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106,184, 267 of SEQ ID NO: 2.

In an embodiment, the variant in the composition of the inventioncomprise substitutions corresponding to one or more of: Q15D, E; G23D,E; T35D, E; T37D, E; N39D, E; A40D, E; P42D, E; N101D, E; S105D, E;G106D, E; F184D, E or T267D, E of SEQ ID NO: 2.

In an embodiment, the amino acid in the parent lipase within 20 angstrom(A) of a cysteine bridge is replaced with a negatively charged aminoacid selected from D or E.

In one aspect, the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 6; SEQ ID NO: 8, SEQ ID NO: 10; or SEQ ID NO: 12.

In an embodiment the composition of the invention comprises a lipasevariant of the parent lipase shown as SEQ ID NO: 2 or of a lipase havingat least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100% sequence identity to theparent lipase and further comprises one or more (e.g. several)substitutions at positions corresponding to amino acid residues 15, 23,35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2.

In an embodiment, the parent lipase has at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4; SEQ ID NO: 6; SEQID NO: 8; SEQ ID NO: 10; or SEQ ID NO: 12.

In an embodiment, the variant of a parent lipase is selected from thegroup consisting of:

(a) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ IDNO: 11, or (ii) the full-length complement of (i);(b) a polypeptide encoded by a polynucleotide having at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to the polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:11; and(c) a fragment of the polypeptide of (a) or (b), in particular of SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; or SEQID NO: 12, which has lipase activity.

In another embodiment, the variant comprised in the composition of theinvention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or moresubstitutions.

In one aspect the invention relates to compositions comprising lipasevariants further comprise one or more (e.g., several) substitutionscorresponding to any of positions selected from: 4, 27, 38, 57, 58, 60,83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 231, 233,249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 2. Inone aspect the invention relates to compositions comprising lipasevariants further comprise one or more (e.g., several) substitutionscorresponding to any of positions selected from: 4V, 27R, 38A, 57G, 58A,60S, 83T, 86V, 91A/N/Q, 94K/R, 97M, 99K, 111A, 150G, 163K, 210K/Q, 216P,225R, 227G, 231R, 233R, 249R, 254S, 255A, 256K/TN, 263Q, 264A, 265T,266D, 267A, and 269N of SEQ ID NO: 2.

In a further embodiment, the variant comprised in the composition of theinvention further comprises a set of substitutions corresponding to thefollowing group (using SEQ ID NO: 2 for numbering) consisting of:

F51V E96V K98E E210K L259F; F51V S85K K98E; F51V K98E E210V; P250V; F51VE87T K98E E210K I252W L259F; F51V L93K K98E; S83T I86P G91A N92D E96LL97P K98Q; F51V N92K K98E; A40E F51V K98E; F51V E87V K98E; Q15R F51VK98E; F51V E87L K98E; F51V K98E L264P; A40Q F51V K98E; F51V K98E L264W;E210R; F51V E56K L69R K98E I196V; Q4K F51V K98E; F51V G91T K98E; A38LF51V K98E; V2K F51V E56K L69R K98E V230R; F51V G91V K98E; F51V E56K L69RE96T K98E; F51V K98E T189D; F51V K98E R125Q; A40I F51V K98E; F51V K98EK223T; R27D A38G E96D A111D K163G S254D T256P; F51V K98E R133Q; F51VL69C K98E; F51V G91A K98E; F51V G91F K98E; F51V K98E S216P; A40G F51VK98E; F51V K98E N251G; F51V G91Y K98E; F51V K98E V154L; G23S F51V K98E;F51V K98E R108K; E1H F51V K98E; F51V K98E S170T; G23T F51V K98E; F51VK98E T267V; F51V K98E H135Y; F51V K98E L269D; F51V E87K K98E E210QL227G; F51V K98E T267L; F51V K98E R139M; R27D F51V K98E; F51V K98EN162G; E210R; F51V L69R E96V K98E E210K F211W; F51V K98E N251G; R27DN33K F51V K98E; G23H N33K F51V K98E; F51V E96T K98E E210K; E87K; F51VE96D K98E E210K; F51V K98E L264E; F51V L69R E96V K98E E210K F211W; F51VE56K K98E K163P V176L E210K Y220F L227G; F51V E210K; V2K F51V L227G;F51V K98I; F51V K98E E210K F211W L259F; F51V E56K L69R G91A N92D E96LK98Q; F51V E56K L69R K98E I196V; F51V E96T K98E E210K; F51V K98E E210KF211W I252W; F51V E87K K98I; F51V K98E N101D K163P E210K; F51V N101DE210K; F51V E96D K98E E210K; F51V K98E V176L E210K L227G; F51V E96T K98EE210K F211W; F51V E56K L69R E96D K98E; F51V E56K L69R K98E T267L; F51VK98E N101D; F51V E56K L69R K98E A180D; F51V E56K L69R E96D K98E; F51VE56K L69R K98E D165N.

In one aspect said variant has increased stability in comparison withthe parent lipase. In one aspect, the stability is stability in thepresence of a source of sulfite, stability in detergent compositions,stability in detergent compositions comprising a source of sulfite,stability under storage conditions, stability under storage conditionsin the presence of a source of sulfite, thermostability, andthermostability in the presence of a source of sulfite.

The non-limiting list of composition components illustrated hereinafterare suitable for use in the compositions and methods herein may bedesirably incorporated in certain aspects of the invention, e.g. toassist or enhance cleaning performance, for treatment of the substrateto be cleaned, or to modify the aesthetics of the composition as is thecase with perfumes, colorants, dyes or the like. The levels of any suchcomponents incorporated in any compositions are in addition to anymaterials previously recited for incorporation. The precise nature ofthese additional components, and levels of incorporation thereof, willdepend on the physical form of the composition and the nature of thecleaning operation for which it is to be used. Although componentsmentioned below are categorized by general header according to aparticular functionality, this is not to be construed as a limitation,as a component may comprise additional functionalities as will beappreciated by the skilled artisan.

Unless otherwise indicated the amounts in percentage is by weight of thecomposition (wt %). Suitable component materials include, but are notlimited to, surfactants, builders, chelating agents, dye transferinhibiting agents, dispersants, enzymes, and enzyme stabilizers,catalytic materials, bleach activators, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal/anti-redeposition agents, brighteners, suds suppressors,dyes, hueing dyes, perfumes, perfume delivery systems, structureelasticizing agents, fabric softeners, carriers, hydrotropes, processingaids, solvents, antioxidants (ex sources of sulfite), bases,preservatives and/or pigments. In addition to the disclosure below,suitable examples of such other components and levels of use are foundin U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348 herebyincorporated by reference.

Thus, in certain aspects the invention do not contain one or more of thefollowing adjuncts materials: surfactants, soaps, builders, chelatingagents, dye transfer inhibiting agents, dispersants, additional enzymes,enzyme stabilizers, catalytic materials, bleach activators, hydrogenperoxide, sources of hydrogen peroxide, preformed peracids, polymericdispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, perfume delivery systems,structure elasticizing agents, fabric softeners, carriers, hydrotropes,processing aids, solvents and/or pigments. However, when one or morecomponents are present, such one or more components may be present asdetailed below:

Surfactants—

The compositions according to the present invention may comprise asurfactant or surfactant system wherein the surfactant can be selectedfrom nonionic surfactants, anionic surfactants, cationic surfactants,ampholytic surfactants, zwitterionic surfactants, semi-polar nonionicsurfactants and mixtures thereof. When present, surfactant is typicallypresent at a level of from 0.1 to 60 wt %, from 0.2 to 40 wt %, from 0.5to 30 wt %, from 1 to 50 wt %, from 1 to 40 wt %, from 1 to 30 wt %,from 1 to 20 wt %, from 3 to 10 wt %, from 3 to 5 wt %, from 5 to 40 wt%, from 5 to 30 wt %, from 5 to 15 wt %, from 3 to 20 wt %, from 3 to 10wt %, from 8 to 12 wt %, from 10 to 12 wt %, from 20 to 25 wt % or from25-60%.

Suitable anionic detersive surfactants include sulphate and sulphonatedetersive surfactants.

Suitable sulphonate detersive surfactants include alkyl benzenesulphonate, in one aspect, C₁₀₋₁₃ alkyl benzene sulphonate. Suitablealkyl benzene sulphonate (LAS) may be obtained, by sulphonatingcommercially available linear alkyl benzene (LAB); suitable LAB includeslow 2-phenyl LAB, such as Isochem® or Petrelab®, other suitable LABinclude high 2-phenyl LAB, such as Hyblene®. A suitable anionicdetersive surfactant is alkyl benzene sulphonate that is obtained byDETAL catalyzed process, although other synthesis routes, such as HF,may also be suitable. In one aspect, a magnesium salt of LAS is used.

Suitable sulphate detersive surfactants include alkyl sulphate, in oneaspect, C₈₋₁₈ alkyl sulphate, or predominantly C₁₂ alkyl sulphate.

Another suitable sulphate detersive surfactant is alkyl alkoxylatedsulphate, in one aspect, alkyl ethoxylated sulphate, in one aspect, aC₈₋₁₈alkyl alkoxylated sulphate, in one aspect, a C₈₋₁₈ alkylethoxylated sulphate, typically the alkyl alkoxylated sulphate has anaverage degree of alkoxylation of from 0.5 to 20, or from 0.5 to 10,typically the alkyl alkoxylated sulphate is a C₈₋₁₈ alkyl ethoxylatedsulphate having an average degree of ethoxylation of from 0.5 to 10,from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3.

The alkyl sulphate, alkyl alkoxylated sulphate and alkyl benzenesulphonates may be linear or branched, substituted or un-substituted.

The detersive surfactant may be a mid-chain branched detersivesurfactant, in one aspect, a mid-chain branched anionic detersivesurfactant, in one aspect, a mid-chain branched alkyl sulphate and/or amid-chain branched alkyl benzene sulphonate, e.g. a mid-chain branchedalkyl sulphate. In one aspect, the mid-chain branches are C₁₋₄ alkylgroups, typically methyl and/or ethyl groups.

Non-limiting examples of anionic surfactants include sulfates andsulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomersof LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates,alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates,alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcoholsulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates(AES or AEOS or FES, also known as alcohol ethoxysulfates or fattyalcohol ether sulfates), secondary alkanesulfonates (SAS), paraffinsulfonates (PS), ester sulfonates, sulfonated fatty acid glycerolesters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES)including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap, andcombinations thereof. Anionic surfactants can be added as correspondingacids or as salts, or can be used as derivatives with ethanolamines, exmonoethanolamine-linear alkylbenzensulfonate (MEA-LAS).

Suitable non-ionic detersive surfactants are selected from the groupconsisting of: C₈-C₁₈ alkyl ethoxylates, such as, NEODOL®; C₆-C₁₂ alkylphenol alkoxylates wherein the alkoxylate units may be ethyleneoxyunits, propyleneoxy units or a mixture thereof; C₁₂-C₁₈ alcohol andC₆-C₁₂ alkyl phenol condensates with ethylene oxide/propylene oxideblock polymers such as Pluronic®; C₁₄-C₂₂ mid-chain branched alcohols;C₁₄-C₂₂ mid-chain branched alkyl alkoxylates, typically having anaverage degree of alkoxylation of from 1 to 30; alkylpolysaccharides, inone aspect, alkylpolyglycosides; polyhydroxy fatty acid amides; ethercapped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

Suitable non-ionic detersive surfactants include alkyl polyglucosideand/or an alkyl alkoxylated alcohol.

In one aspect, non-ionic detersive surfactants include alkyl alkoxylatedalcohols, in one aspect C₈₋₁₈ alkyl alkoxylated alcohol, e.g. a C₈₋₁₈alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol may have anaverage degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may bea C₈₋₁₈ alkyl ethoxylated alcohol having an average degree ofethoxylation of from 1 to 10, from 1 to 7, more from 1 to 5 or from 3 to7. The alkyl alkoxylated alcohol can be linear or branched, andsubstituted or un-substituted. Suitable nonionic surfactants includeLutensol®.

Non-limiting examples of nonionic surfactants include alcoholethoxylates (AE or AEO), alcohol propoxylates, propoxylated fattyalcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylatedand/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates(APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG),alkoxylated amines, fatty acid monoethanolamides (FAM), fatty aciddiethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM),propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fattyacid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides,GA, or fatty acid glucamides, FAGA), methyl ester ethoxylates (MEE), aswell as products available under the trade names SPAN and TWEEN, andcombinations thereof.

Suitable cationic detersive surfactants include alkyl pyridiniumcompounds, alkyl quaternary ammonium compounds, alkyl quaternaryphosphonium compounds, alkyl ternary sulphonium compounds, and mixturesthereof.

Suitable cationic detersive surfactants are quaternary ammoniumcompounds having the general formula: (R)(R₁)(R₂)(R₃)N⁺X⁻, wherein, R isa linear or branched, substituted or unsubstituted C₆₋₁₈ alkyl oralkenyl moiety, R₁ and R₂ are independently selected from methyl orethyl moieties, R₃ is a hydroxyl, hydroxymethyl or a hydroxyethylmoiety, X is an anion which provides charge neutrality, suitable anionsinclude: halides, e.g. chloride; sulphate; and sulphonate. Suitablecationic detersive surfactants are mono-C₆₋₁₈ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chlorides. Highly suitable cationicdetersive surfactants are mono-C₈₋₁₀ alkyl mono-hydroxyethyl di-methylquaternary ammonium chloride, mono-C₁₀₋₁₂ alkyl mono-hydroxyethyldi-methyl quaternary ammonium chloride and mono-C₁₀ alkylmono-hydroxyethyl di-methyl quaternary ammonium chloride.

Non-limiting examples of cationic surfactants includealkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide(CTAB), dimethyldistearylammonium chloride (DSDMAC), andalkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,alkoxylated quaternary ammonium (AQA) compounds, ester quats, andcombinations thereof.

Suitable amphoteric/zwitterionic surfactants include amine oxides andbetaines such as alkyldimethylbetaines, sulfobetaines, or combinationsthereof. Amine-neutralized anionic surfactants—Anionic surfactants ofthe present invention and adjunct anionic cosurfactants, may exist in anacid form, and said acid form may be neutralized to form a surfactantsalt which is desirable for use in the present detergent compositions.Typical agents for neutralization include the metal counterion base suchas hydroxides, eg, NaOH or KOH. Further preferred agents forneutralizing anionic surfactants of the present invention and adjunctanionic surfactants or cosurfactants in their acid forms includeammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitablenon-limiting examples including monoethanolamine, diethanolamine,triethanolamine, and other linear or branched alkanolamines known in theart; e.g., highly preferred alkanolamines include 2-amino-1-propanol,1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amineneutralization may be done to a full or partial extent, e.g. part of theanionic surfactant mix may be neutralized with sodium or potassium andpart of the anionic surfactant mix may be neutralized with amines oralkanolamines.

Non-limiting examples of semipolar surfactants include amine oxides (AO)such as alkyldimethylamineoxide

Surfactant systems comprising mixtures of one or more anionic and inaddition one or more nonionic surfactants optionally with an additionalsurfactant such as a cationic surfactant, may be preferred. Preferredweight ratios of anionic to nonionic surfactant are at least 2:1, or atleast 1:1 to 1:10.

In one aspect, a surfactant system may comprise a mixture of isoprenoidsurfactants represented by formula A and formula B:

where Y is CH₂ or null, and Z may be chosen such that the resultingsurfactant is selected from the following surfactants: an alkylcarboxylate surfactant, an alkyl polyalkoxy surfactant, an alkyl anionicpolyalkoxy sulfate surfactant, an alkyl glycerol ester sulfonatesurfactant, an alkyl dimethyl amine oxide surfactant, an alkylpolyhydroxy based surfactant, an alkyl phosphate ester surfactant, analkyl glycerol sulfonate surfactant, an alkyl polygluconate surfactant,an alkyl polyphosphate ester surfactant, an alkyl phosphonatesurfactant, an alkyl polyglycoside surfactant, an alkyl monoglycosidesurfactant, an alkyl diglycoside surfactant, an alkyl sulfosuccinatesurfactant, an alkyl disulfate surfactant, an alkyl disulfonatesurfactant, an alkyl sulfosuccinamate surfactant, an alkyl glucamidesurfactant, an alkyl taurinate surfactant, an alkyl sarcosinatesurfactant, an alkyl glycinate surfactant, an alkyl isethionatesurfactant, an alkyl dialkanolamide surfactant, an alkylmonoalkanolamide surfactant, an alkyl monoalkanolamide sulfatesurfactant, an alkyl diglycolamide surfactant, an alkyl diglycolamidesulfate surfactant, an alkyl glycerol ester surfactant, an alkylglycerol ester sulfate surfactant, an alkyl glycerol ether surfactant,an alkyl glycerol ether sulfate surfactant, alkyl methyl ester sulfonatesurfactant, an alkyl polyglycerol ether surfactant, an alkylpolyglycerol ether sulfate surfactant, an alkyl sorbitan estersurfactant, an alkyl ammonioalkanesulfonate surfactant, an alkylamidopropyl betaine surfactant, an alkyl allylated quat basedsurfactant, an alkyl monohydroxyalkyl-di-alkylated quat basedsurfactant, an alkyl di-hydroxyalkyl monoalkyl quat based surfactant, analkylated quat surfactant, an alkyl trimethylammonium quat surfactant,an alkyl polyhydroxalkyl oxypropyl quat based surfactant, an alkylglycerol ester quat surfactant, an alkyl glycol amine quat surfactant,an alkyl monomethyl dihydroxyethyl quaternary ammonium surfactant, analkyl dimethyl monohydroxyethyl quaternary ammonium surfactant, an alkyltrimethylammonium surfactant, an alkyl imidazoline-based surfactant, analken-2-yl-succinate surfactant, an alkyl a-sulfonated carboxylic acidsurfactant, an alkyl a-sulfonated carboxylic acid alkyl estersurfactant, an alpha olefin sulfonate surfactant, an alkyl phenolethoxylate surfactant, an alkyl benzenesulfonate surfactant, an alkylsulfobetaine surfactant, an alkyl hydroxysulfobetaine surfactant, analkyl ammoniocarboxylate betaine surfactant, an alkyl sucrose estersurfactant, an alkyl alkanolamide surfactant, an alkyldi(polyoxyethylene) monoalkyl ammonium surfactant, an alkylmono(polyoxyethylene) dialkyl ammonium surfactant, an alkyl benzyldimethylammonium surfactant, an alkyl aminopropionate surfactant, analkyl amidopropyl dimethylamine surfactant, or a mixture thereof; and ifZ is a charged moiety, Z is charge-balanced by a suitable metal ororganic counter ion. Suitable counter ions include a metal counter ion,an amine, or an alkanolamine, e.g., C1-C6 alkanolammonium. Morespecifically, suitable counter ions include Na+, Ca+, Li+, K+, Mg+,e.g., monoethanolamine (MEA), diethanolamine (DEA), triethanolamine(TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine,dimethylethanolamine, monoisopropanolamine, triisopropanolamine,I-amino-3-propanol, or mixtures thereof. In one aspect, the compositionscontain from 5% to 97% of one or more non-isoprenoid surfactants; andone or more adjunct cleaning additives; wherein the weight ratio ofsurfactant of formula A to surfactant of formula B is from 50:50 to95:5.

Soap—

The compositions herein may contain soap. Without being limited bytheory, it may be desirable to include soap as it acts in part as asurfactant and in part as a builder and may be useful for suppression offoam and may furthermore interact favorably with the various cationiccompounds of the composition to enhance softness on textile fabricstreaded with the inventive compositions. Any soap known in the art foruse in laundry detergents may be utilized. In one aspect, thecompositions contain from 0 wt % to 20 wt %, from 0.5 wt % to 20 wt %,from 4 wt % to 10 wt %, or from 4 wt % to 7 wt % of soap. In one aspect,the compositions contain from 0 wt % to 5 wt %; from 0 wt % to 2 wt %,or from 0 wt % to 1 wt %.

Examples of soap useful herein include oleic acid soaps, palmitic acidsoaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soapsare in the form of mixtures of fatty acid soaps having different chainlengths and degrees of substitution. One such mixture is topped palmkernel fatty acid.

In one aspect, the soap is selected from free fatty acid. Suitable fattyacids are saturated and/or unsaturated and can be obtained from naturalsources such a plant or animal esters (e.g., palm kernel oil, palm oil,coconut oil, babassu oil, safflower oil, tall oil, castor oil, tallowand fish oils, grease, and mixtures thereof), or synthetically prepared(e.g., via the oxidation of petroleum or by hydrogenation of carbonmonoxide via the Fisher Tropsch process).

Examples of suitable saturated fatty acids for use in the compositionsof this invention include captic, lauric, myristic, palmitic, stearic,arachidic and behenic acid. Suitable unsaturated fatty acid speciesinclude: palmitoleic, oleic, linoleic, linolenic and ricinoleic acid.Examples of preferred fatty acids are saturated Cn fatty acid, saturatedCi₂-Ci₄ fatty acids, and saturated or unsaturated Cn to Ci₈ fatty acids,and mixtures thereof.

When present, the weight ratio of fabric softening cationic cosurfactantto fatty acid is preferably from about 1:3 to about 3:1, more preferablyfrom about 1:1.5 to about 1.5:1, most preferably about 1:1.

Levels of soap and of nonsoap anionic surfactants herein are percentagesby weight of the detergent composition, specified on an acid form basis.However, as is commonly understood in the art, anionic surfactants andsoaps are in practice neutralized using sodium, potassium oralkanolammonium bases, such as sodium hydroxide or monoethanolamine.

Hydrotropes—

The compositions of the present invention may comprise one or morehydrotropes. A hydrotrope is a compound that solubilises hydrophobiccompounds in aqueous solutions (or oppositely, polar substances in anon-polar environment). Typically, hydrotropes have both hydrophilic anda hydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscontaining substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain from 0 to 10 wt %, such as from 0 to 5 wt %,0.5 to 5 wt %, or from 3% to 5 wt %, of a hydrotrope. Any hydrotropeknown in the art for use in detergents may be utilized.

Non-limiting examples of hydrotropes include sodium benzenesulfonate,sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodiumcumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcoholsand polyglycolethers, sodium hydroxynaphthoate, sodiumhydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof.

Builders—

The compositions of the present invention may comprise one or morebuilders, co-builders, builder systems or a mixture thereof. When abuilder is used, the cleaning composition will typically comprise from 0to 65 wt %, at least 1 wt %, from 2 to 60 wt % or from 5 to 10 wt %builder. In a dish wash cleaning composition, the level of builder istypically 40 to 65 wt % or 50 to 65 wt %. The composition may besubstantially free of builder; substantially free means “no deliberatelyadded” zeolite and/or phosphate. Typical zeolite builders includezeolite A, zeolite P and zeolite MAP. A typical phosphate builder issodium tri-polyphosphate.

The builder and/or co-builder may particularly be a chelating agent thatforms water-soluble complexes with Ca and Mg. Any builder and/orco-builder known in the art for use in detergents may be utilized.Non-limiting examples of builders include zeolites, diphosphates(pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such assodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst),ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), andcombinations thereof.

The cleaning composition may include a co-builder alone, or incombination with a builder, e.g. a zeolite builder. Non-limitingexamples of co-builders include homopolymers of polyacrylates orcopolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylicacid/maleic acid) (PAA/PMA). Further non-limiting examples includecitrate, chelators such as aminocarboxylates, aminopolycarboxylates andphosphonates, and alkyl- or alkenylsuccinic acid. Additional specificexamples include 2,2′,2″-nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinicacid (EDDS), methylglycinediacetic acid (MGDA), glutamicacid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonicacid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonicacid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonicacid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), asparticacid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA),aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid(SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA),α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA),isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N—(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine(DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP),aminotris(methylenephosphonic acid) (ATMP), and combinations and saltsthereof. Further exemplary builders and/or co-builders are described in,e.g., WO09/102854, U.S. Pat. No. 5,977,053.

In one aspect, the invention relates to compositions comprising a lipasevariant of a parent lipase, wherein the variant has lipase activity,comprises a substitution at a position corresponding to position 15, 23,35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of the parent lipase, thecomposition comprising up to 10 wt % or 15 wt % aluminosilicate(anhydrous basis) and/or phosphate builder, the composition having areserve alkalinity of greater than 4 or 7.5.

In one aspect the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 6; SEQ ID NO: 8, SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect thesubstitutions are selected from positions Q15D,E; G23,D,E; T35D,E;T37D,E; N39D,E; A40D,E; P42D,E; N101D,E; S105D,E; G106D,E; F184D,E orT267D,E of the parent lipase, in particular SEQ ID NO: 2.

As used herein the term “reserve alkalinity” is a measure of thebuffering capacity of the composition (g/NaOH/100 g composition)determined by titrating a 1% (w/v) solution of composition withhydrochloric acid to pH 7.5 i.e. in order to calculate reservealkalinity. Reserve alkalinity may be calculated as disclosed on page 9in WO2006/090335.

In one aspect the invention relates to compositions comprising lipasevariants further comprise one or more (e.g., several) substitutionscorresponding to any of positions selected from: 4, 27, 38, 57, 58, 60,83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 230, 231,233, 249, 250, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ IDNO: 12. In one aspect the invention relates to compositions comprisinglipase variants further comprise one or more (e.g., several)substitutions corresponding to any of positions selected from: 4V, 27R,38A, 57G, 58A, 60S, 83T, 86V, 91A/N/Q, 94K/R, 97M, 99K, 111A, 150G,163K, 210K/Q, 216P, 225R, 227G, 231R, 233R, 249R, 254S, 255A, 256K/TNV,263Q, 264A, 265T, 266D, 267A, and 269N of SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12.

Chelating Agents and Crystal Growth Inhibitors—

The compositions herein may contain a chelating agent and/or a crystalgrowth inhibitor. Suitable molecules include copper, iron and/ormanganese chelating agents and mixtures thereof. Suitable moleculesinclude DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethanediphosphonic acid), DTPMP (Diethylene triamine penta(methylenephosphonic acid)), 1,2-Dihydroxybenzene-3,5-disulfonic acid disodiumsalt hydrate, ethylenediamine, diethylene triamine,ethylenediaminedisuccinic acid (EDDS), ethylenediaminetetraacetic acid(EDTA), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin (CMI)and 2-Phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM) andderivatives thereof. Typically, the composition may comprise from 0.005to 15 wt % or from 3.0 to 10 wt % chelating agent or crystal growthinhibitor.

Bleach Component—

The bleach component suitable for incorporation in the methods andcompositions of the invention comprise one or a mixture of more than onebleach component. Suitable bleach components include bleachingcatalysts, photobleaches, bleach activators, hydrogen peroxide, sourcesof hydrogen peroxide, pre-formed peracids and mixtures thereof. Ingeneral, when a bleach component is used, the compositions of thepresent invention may comprise from 0 to 30 wt %, from 0.00001 to 90 wt%, 0.0001 to 50 wt %, from 0.001 to 25 wt % or from 1 to 20 wt %.Examples of suitable bleach components include:

(1) Pre-formed peracids: Suitable preformed peracids include, but arenot limited to, compounds selected from the group consisting ofpre-formed peroxyacids or salts thereof, typically either aperoxycarboxylic acid or salt thereof, or a peroxysulphonic acid or saltthereof.

The pre-formed peroxyacid or salt thereof is preferably aperoxycarboxylic acid or salt thereof, typically having a chemicalstructure corresponding to the following chemical formula:

wherein: R¹⁴ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁴ group can be linear or branched,substituted or unsubstituted; and Y is any suitable counter-ion thatachieves electric charge neutrality, preferably Y is selected fromhydrogen, sodium or potassium. Preferably, R¹⁴ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably, the peroxyacid orsalt thereof is selected from peroxyhexanoic acid, peroxyheptanoic acid,peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any saltthereof, or any combination thereof. Particularly preferred peroxyacidsare phthalimido-peroxy-alkanoic acids, in particular ε-phthahlimidoperoxy hexanoic acid (PAP). Preferably, the peroxyacid or salt thereofhas a melting point in the range of from 30° C. to 60° C.

The pre-formed peroxyacid or salt thereof can also be a peroxysulphonicacid or salt thereof, typically having a chemical structurecorresponding to the following chemical formula:

wherein: R¹⁵ is selected from alkyl, aralkyl, cycloalkyl, aryl orheterocyclic groups; the R¹⁵ group can be linear or branched,substituted or unsubstituted; and Z is any suitable counter-ion thatachieves electric charge neutrality, preferably Z is selected fromhydrogen, sodium or potassium. Preferably R¹⁵ is a linear or branched,substituted or unsubstituted C₆₋₉ alkyl. Preferably such bleachcomponents may be present in the compositions of the invention in anamount from 0.01 to 50 wt % or from 0.1 to 20 wt %.

(2) Sources of hydrogen peroxide include e.g., inorganic perhydratesalts, including alkali metal salts such as sodium salts of perborate(usually mono- or tetra-hydrate), percarbonate, persulphate,perphosphate, persilicate salts and mixtures thereof. In one aspect ofthe invention the inorganic perhydrate salts such as those selected fromthe group consisting of sodium salts of perborate, percarbonate andmixtures thereof. When employed, inorganic perhydrate salts aretypically present in amounts of 0.05 to 40 wt % or 1 to 30 wt % of theoverall composition and are typically incorporated into suchcompositions as a crystalline solid that may be coated. Suitablecoatings include: inorganic salts such as alkali metal silicate,carbonate or borate salts or mixtures thereof, or organic materials suchas water-soluble or dispersible polymers, waxes, oils or fatty soaps.Preferably such bleach components may be present in the compositions ofthe invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

(3) The term bleach activator is meant herein as a compound which reactswith hydrogen peroxide to form a peracid via perhydrolysis. The peracidthus formed constitutes the activated bleach. Suitable bleach activatorsto be used herein include those belonging to the class of esters,amides, imides or anhydrides. Suitable bleach activators are thosehaving R—(C═O)-L wherein R is an alkyl group, optionally branched,having, when the bleach activator is hydrophobic, from 6 to 14 carbonatoms, or from 8 to 12 carbon atoms and, when the bleach activator ishydrophilic, less than 6 carbon atoms or less than 4 carbon atoms; and Lis leaving group. Examples of suitable leaving groups are benzoic acidand derivatives thereof—especially benzene sulphonate. Suitable bleachactivators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzenesulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED),sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS orDOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those disclosedin WO98/17767. A family of bleach activators is disclosed in EP624154and particularly preferred in that family is acetyl triethyl citrate(ATC). ATC or a short chain triglyceride like triacetin has theadvantage that it is environmentally friendly. Furthermore, acetyltriethyl citrate and triacetin have good hydrolytical stability in theproduct upon storage and are efficient bleach activators. Finally, ATCis multifunctional, as the citrate released in the perhydrolysisreaction may function as a builder. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthalimido)peroxyhexanoic acid (PAP). Suitable bleach activators arealso disclosed in WO98/17767. While any suitable bleach activator may beemployed, in one aspect of the invention the subject cleaningcomposition may comprise NOBS, TAED or mixtures thereof. When present,the peracid and/or bleach activator is generally present in thecomposition in an amount of 0.1 to 60 wt %, 0.5 to 40 wt % or 0.6 to 10wt % based on the fabric and home care composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof. Preferablysuch bleach components may be present in the compositions of theinvention in an amount of 0.01 to 50 wt %, or 0.1 to 20 wt %.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

(4) Diacyl peroxides—preferred diacyl peroxide bleaching species includethose selected from diacyl peroxides of the general formula:R¹—C(O)—OO—(O)C—R², in which R¹ represents a C₆-C₁₈ alkyl, preferablyC₆-C₁₂ alkyl group containing a linear chain of at least 5 carbon atomsand optionally containing one or more substituents (e.g. —N′ (CH₃)₃,—COOH or —CN) and/or one or more interrupting moieties (e.g. —CONH— or—CH═CH—) interpolated between adjacent carbon atoms of the alkylradical, and R² represents an aliphatic group compatible with a peroxidemoiety, such that R¹ and R² together contain a total of 8 to 30 carbonatoms. In one preferred aspect R¹ and R² are linear unsubstituted C₆-C₁₂alkyl chains. Most preferably R¹ and R² are identical. Diacyl peroxides,in which both R¹ and R² are C₆-C₁₂ alkyl groups, are particularlypreferred. Preferably, at least one of, most preferably only one of, theR groups (R₁ or R₂), does not contain branching or pendant rings in thealpha position, or preferably neither in the alpha nor beta positions ormost preferably in none of the alpha or beta or gamma positions. In onefurther preferred aspect, the DAP may be asymmetric, such thatpreferably the hydrolysis of R1 acyl group is rapid to generate peracid,but the hydrolysis of R2 acyl group is slow.

The tetraacyl peroxide bleaching species is preferably selected fromtetraacyl peroxides of the general formula:R³—C(O)—OO—C(O)—(CH₂)n-C(O)—OO—C(O)—R³, in which R³ represents a C₁-C₉alkyl, or C₃-C₇, group and n represents an integer from 2 to 12, or 4 to10 inclusive.

Preferably, the diacyl and/or tetraacyl peroxide bleaching species ispresent in an amount sufficient to provide at least 0.5 ppm, at least 10ppm, or at least 50 ppm by weight of the wash liquor. In a preferredaspect, the bleaching species is present in an amount sufficient toprovide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the washliquor.

Preferably the bleach component comprises a bleach catalyst (5 and 6).

(5) Preferred are organic (non-metal) bleach catalysts include bleachcatalyst capable of accepting an oxygen atom from a peroxyacid and/orsalt thereof, and transferring the oxygen atom to an oxidizeablesubstrate. Suitable bleach catalysts include, but are not limited to:iminium cations and polyions; iminium zwitterions; modified amines;modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones andmixtures thereof.

Suitable iminium cations and polyions include, but are not limited to,N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared asdescribed in Tetrahedron (1992), 49(2), 423-38 (e.g. compound 4, p.433); N-methyl-3,4-dihydroisoquinolinium p-toluene sulphonate, preparedas described in U.S. Pat. No. 5,360,569 (e.g. Column 11, Example 1); andN-octyl-3,4-dihydroisoquinolinium p-toluene sulphonate, prepared asdescribed in U.S. Pat. No. 5,360,568 (e.g. Column 10, Ex. 3).

Suitable iminium zwitterions include, but are not limited to,N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared asdescribed in U.S. Pat. No. 5,576,282 (e.g. Column 31, Ex. II);N-[2-(sulphooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, preparedas described in U.S. Pat. No. 5,817,614 (e.g. Column 32, Ex. V);2-[3-[(2-ethylhexyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt, prepared as described in WO05/047264 (e.g. p. 18, Ex. 8),and2-[3-[(2-butyloctyl)oxy]-2-(sulphooxy)propyl]-3,4-dihydroisoquinolinium,inner salt.

Suitable modified amine oxygen transfer catalysts include, but are notlimited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can bemade according to the procedures described in Tetrahedron Letters(1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfercatalysts include, but are not limited to, sodium1-hydroxy-N-oxy-N-[2-(sulphooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

Suitable N-sulphonyl imine oxygen transfer catalysts include, but arenot limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, preparedaccording to the procedure described in the Journal of Organic Chemistry(1990), 55(4), 1254-61.

Suitable N-phosphonyl imine oxygen transfer catalysts include, but arenot limited to,[R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinicamide, which can be made according to the procedures described in theJournal of the Chemical Society, Chemical Communications (1994), (22),2569-70.

Suitable N-acyl imine oxygen transfer catalysts include, but are notlimited to, [N(E)]-N-(phenylmethylene)acetamide, which can be madeaccording to the procedures described in Polish Journal of Chemistry(2003), 77(5), 577-590.

Suitable thiadiazole dioxide oxygen transfer catalysts include but arenot limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, whichcan be made according to the procedures described in U.S. Pat. No.5,753,599 (Column 9, Ex. 2).

Suitable perfluoroimine oxygen transfer catalysts include, but are notlimited to,(Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride,which can be made according to the procedures described in TetrahedronLetters (1994), 35(34), 6329-30.

Suitable cyclic sugar ketone oxygen transfer catalysts include, but arenot limited to,1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose asprepared in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

Preferably, the bleach catalyst comprises an iminium and/or carbonylfunctional group and is typically capable of forming an oxaziridiniumand/or dioxirane functional group upon acceptance of an oxygen atom,especially upon acceptance of an oxygen atom from a peroxyacid and/orsalt thereof. Preferably, the bleach catalyst comprises an oxaziridiniumfunctional group and/or is capable of forming an oxaziridiniumfunctional group upon acceptance of an oxygen atom, especially uponacceptance of an oxygen atom from a peroxyacid and/or salt thereof.Preferably, the bleach catalyst comprises a cyclic iminium functionalgroup, preferably wherein the cyclic moiety has a ring size of from fiveto eight atoms (including the nitrogen atom), preferably six atoms.Preferably, the bleach catalyst comprises an aryliminium functionalgroup, preferably a bi-cyclic aryliminium functional group, preferably a3,4-dihydroisoquinolinium functional group. Typically, the iminefunctional group is a quaternary imine functional group and is typicallycapable of forming a quaternary oxaziridinium functional group uponacceptance of an oxygen atom, especially upon acceptance of an oxygenatom from a peroxyacid and/or salt thereof. In one aspect, the detergentcomposition comprises a bleach component having a log P_(o/w) no greaterthan 0, no greater than −0.5, no greater than −1.0, no greater than−1.5, no greater than −2.0, no greater than −2.5, no greater than −3.0,or no greater than −3.5. The method for determining log P_(o/w) isdescribed in more detail below.

Typically, the bleach ingredient is capable of generating a bleachingspecies having a X_(so) of from 0.01 to 0.30, from 0.05 to 0.25, or from0.10 to 0.20. The method for determining X_(so) is described in moredetail below. For example, bleaching ingredients having anisoquinolinium structure are capable of generating a bleaching speciesthat has an oxaziridinium structure. In this example, the X_(so) is thatof the oxaziridinium bleaching species.

Preferably, the bleach catalyst has a chemical structure correspondingto the following chemical formula:

wherein: n and m are independently from 0 to 4, preferably n and m areboth 0; each R¹ is independently selected from a substituted orunsubstituted radical selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, fused aryl, heterocyclic ring, fusedheterocyclic ring, nitro, halo, cyano, sulphonato, alkoxy, keto,carboxylic, and carboalkoxy radicals; and any two vicinal R¹substituents may combine to form a fused aryl, fused carbocyclic orfused heterocyclic ring; each R² is independently selected from asubstituted or unsubstituted radical independently selected from thegroup consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl,aralkyl, alkylenes, heterocyclic ring, alkoxys, arylcarbonyl groups,carboxyalkyl groups and amide groups; any R² may be joined together withany other of R² to form part of a common ring; any geminal R² maycombine to form a carbonyl; and any two R² may combine to form asubstituted or unsubstituted fused unsaturated moiety; R³ is a C₁ to C₂₀substituted or unsubstituted alkyl; R⁴ is hydrogen or the moietyQ_(t)-A, wherein: Q is a branched or unbranched alkylene, t=0 or 1 and Ais an anionic group selected from the group consisting of OSO₃ ⁻, SO₃ ⁻,CO₂ ⁻, OCO₂ ⁻, OPO₃ ²⁻, OPO₃H⁻ and OPO₂ ⁻; R⁵ is hydrogen or the moiety—CR¹¹R¹²—Y-G_(b)-Y_(c)—[(CR⁹R¹⁰)_(y)—O]_(k)—R⁸, wherein: each Y isindependently selected from the group consisting of O, S, N—H, or N—R⁸;and each R⁸ is independently selected from the group consisting ofalkyl, aryl and heteroaryl, said moieties being substituted orunsubstituted, and whether substituted or unsubsituted said moietieshaving less than 21 carbons; each G is independently selected from thegroup consisting of CO, SO₂, SO, PO and PO₂; R⁹ and R¹⁰ areindependently selected from the group consisting of H and C₁-C₄ alkyl;R¹¹ and R¹² are independently selected from the group consisting of Hand alkyl, or when taken together may join to form a carbonyl; b=0 or 1;c can=0 or 1, but c must=0 if b=0; y is an integer from 1 to 6; k is aninteger from 0 to 20; R⁶ is H, or an alkyl, aryl or heteroaryl moiety;said moieties being substituted or unsubstituted; and X, if present, isa suitable charge balancing counterion, preferably X is present when R⁴is hydrogen, suitable X, include but are not limited to: chloride,bromide, sulphate, methosulphate, sulphonate, p-toluenesulphonate,borontetraflouride and phosphate.In one aspect of the present invention, the bleach catalyst has astructure corresponding to general formula below:

wherein R¹³ is a branched alkyl group containing from three to 24 carbonatoms (including the branching carbon atoms) or a linear alkyl groupcontaining from one to 24 carbon atoms; preferably R¹³ is a branchedalkyl group containing from eight to 18 carbon atoms or linear alkylgroup containing from eight to eighteen carbon atoms; preferably R¹³ isselected from the group consisting of 2-propylheptyl, 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl,n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl;preferably R¹³ is selected from the group consisting of 2-butyloctyl,2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

Preferably the bleach component comprises a source of peracid inaddition to bleach catalyst, particularly organic bleach catalyst. Thesource of peracid may be selected from (a) pre-formed peracid; (b)percarbonate, perborate or persulfate salt (hydrogen peroxide source)preferably in combination with a bleach activator; and (c) perhydrolaseenzyme and an ester for forming peracid in situ in the presence of waterin a textile or hard surface treatment step.

When present, the peracid and/or bleach activator is generally presentin the composition in an amount of from 0.1 to 60 wt %, from 0.5 to 40wt % or from 0.6 to 10 wt % based on the composition. One or morehydrophobic peracids or precursors thereof may be used in combinationwith one or more hydrophilic peracid or precursor thereof.

The amounts of hydrogen peroxide source and peracid or bleach activatormay be selected such that the molar ratio of available oxygen (from theperoxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

(6) Metal-containing Bleach Catalysts—The bleach component may beprovided by a catalytic metal complex. One type of metal-containingbleach catalyst is a catalyst system comprising a transition metalcation of defined bleach catalytic activity, such as copper, iron,titanium, ruthenium, tungsten, molybdenum, or manganese cations, anauxiliary metal cation having little or no bleach catalytic activity,such as zinc or aluminum cations, and a sequestrate having definedstability constants for the catalytic and auxiliary metal cations,particularly ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid) and water-soluble saltsthereof. Such catalysts are disclosed in U.S. Pat. No. 4,430,243.Preferred catalysts are described in WO09/839406, U.S. Pat. No.6,218,351 and WO00/012667. Particularly preferred are transition metalcatalyst or ligands therefore that are cross-bridged polydentate N-donorligands.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, e.g., the manganese-based catalysts disclosed inU.S. Pat. No. 5,576,282.

Cobalt bleach catalysts useful herein are known, and are described e.g.in U.S. Pat. Nos. 5,597,936; 5,595,967. Such cobalt catalysts arereadily prepared by known procedures, such as taught e.g. in U.S. Pat.Nos. 5,597,936 and 5,595,967.

Compositions herein may also suitably include a transition metal complexof ligands such as bispidones (U.S. Pat. No. 7,501,389) and/ormacropolycyclic rigid ligands—abbreviated as “MRLs”. As a practicalmatter, and not by way of limitation, the compositions and processesherein can be adjusted to provide on the order of at least one part perhundred million of the active MRL species in the aqueous washing medium,and will typically provide from 0.005 to 25 ppm, from 0.05 to 10 ppm, orfrom 0.1 to 5 ppm, of the MRL in the wash liquor.

Suitable transition-metals in the instant transition-metal bleachcatalyst include e.g. manganese, iron and chromium. Suitable MRLsinclude 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitabletransition metal MRLs are readily prepared by known procedures, such astaught e.g. in U.S. Pat. No. 6,225,464 and WO00/32601.

(7) Photobleaches—suitable photobleaches include e.g. sulfonated zincphthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes andmixtures thereof. Preferred bleach components for use in the presentcompositions of the invention comprise a hydrogen peroxide source,bleach activator and/or organic peroxyacid, optionally generated in situby the reaction of a hydrogen peroxide source and bleach activator, incombination with a bleach catalyst. Preferred bleach components comprisebleach catalysts, preferably organic bleach catalysts, as describedabove.

Particularly preferred bleach components are the bleach catalysts inparticular the organic bleach catalysts.

Exemplary bleaching systems are also described, e.g. in WO2007/087258,WO2007/087244, WO2007/087259 and WO2007/087242.

Fabric Hueing Agents—

The composition may comprise a fabric hueing agent. Suitable fabrichueing agents include dyes, dye-clay conjugates, and pigments. Suitabledyes include small molecule dyes and polymeric dyes. Suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of dyes falling into the Color Index (C.I.) classificationsof Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, AcidViolet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof.

In one aspect, suitable small molecule dyes include small molecule dyesselected from the group consisting of Color Index (Society of Dyers andColorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35,Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99,Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, AcidViolet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, AcidBlue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, AcidBlue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3,Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, BasicBlue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75,Basic Blue 159 and mixtures thereof. In one aspect, suitable smallmolecule dyes include small molecule dyes selected from the groupconsisting of Color Index (Society of Dyers and Colorists, Bradford, UK)numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, AcidRed 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, AcidBlue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51and mixtures thereof. In one aspect, suitable small molecule dyesinclude small molecule dyes selected from the group consisting of ColorIndex (Society of Dyers and Colorists, Bradford, UK) numbers Acid Violet17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, AcidRed 150, Acid Blue 29, Acid Blue 113 or mixtures thereof.

Suitable polymeric dyes include polymeric dyes selected from the groupconsisting of polymers containing conjugated chromogens (dye-polymerconjugates) and polymers with chromogens co-polymerized into thebackbone of the polymer and mixtures thereof.

In one aspect, suitable polymeric dyes include polymeric dyes selectedfrom the group consisting of fabric-substantive colorants sold under thename of Liquitint® (Milliken), dye-polymer conjugates formed from atleast one reactive dye and a polymer selected from the group consistingof polymers comprising a moiety selected from the group consisting of ahydroxyl moiety, a primary amine moiety, a secondary amine moiety, athiol moiety and mixtures thereof. In still one aspect, suitablepolymeric dyes include polymeric dyes selected from the group consistingof Liquitint® Violet CT, carboxymethyl cellulose (CMC) conjugated with areactive blue, reactive violet or reactive red dye such as CMCconjugated with C.I. Reactive Blue 19, sold by Megazyme, Wicklow,Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC,alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophenepolymeric colorants, and mixtures thereof.

Preferred hueing dyes include the whitening agents found in WO008/87497.These whitening agents may be characterized by the following structure(I):

wherein R₁ and R₂ can independently be selected from:

a) [(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

b) R₁=alkyl, aryl or aryl alkyl and R₂═[(CH₂CR′HO)_(x)(CH₂CR″HO)_(y)H]

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

c) R₁═[CH₂CH₂(OR₃)CH₂OR₄] and R₂═[CH₂CH₂(O R₃)CH₂O R₄]

wherein R₃ is selected from the group consisting of H, (CH₂CH₂O)_(z)H,and mixtures thereof; and wherein z=0 to 10;

wherein R₄ is selected from the group consisting of (C₁-C₁₆)alkyl, arylgroups, and mixtures thereof; and

d) wherein R1 and R2 can independently be selected from the aminoaddition product of styrene oxide, glycidyl methyl ether, isobutylglycidyl ether, isopropylglycidyl ether, t-butyl glycidyl ether,2-ethylhexylgycidyl ether, and glycidylhexadecyl ether, followed by theaddition of from 1 to 10 alkylene oxide units.

A preferred whitening agent of the present invention may becharacterized by the following structure (II):

wherein R′ is selected from the group consisting of H, CH₃,CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof; wherein R″ is selected fromthe group consisting of H, CH₂O(CH₂CH₂O)_(z)H, and mixtures thereof;wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

A further preferred whitening agent of the present invention may becharacterized by the following structure (III):

typically comprising a mixture having a total of 5 EO groups. Suitablepreferred molecules are those in Structure I having the followingpendant groups in “part a” above.

TABLE 1 R1 R2 R′ R″ X y R′ R″ X y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c= b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1Further whitening agents of use include those described in US2008/34511(Unilever). A preferred agent is “Violet 13”.

Suitable dye clay conjugates include dye clay conjugates selected fromthe group comprising at least one cationic/basic dye and a smectiteclay, and mixtures thereof. In one aspect, suitable dye clay conjugatesinclude dye clay conjugates selected from the group consisting of onecationic/basic dye selected from the group consisting of C.I. BasicYellow 1 through 108, C.I. Basic Orange 1 through 69, C.I. Basic Red 1through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue 1 through164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through 23, CIBasic Black 1 through 11, and a clay selected from the group consistingof Montmorillonite clay, Hectorite clay, Saponite clay and mixturesthereof. In still one aspect, suitable dye clay conjugates include dyeclay conjugates selected from the group consisting of: MontmorilloniteBasic Blue B7 C.I. 42595 conjugate, Montmorillonite Basic Blue B9 C.I.52015 conjugate, Montmorillonite Basic Violet V3 C.I. 42555 conjugate,Montmorillonite Basic Green G1 C.I. 42040 conjugate, MontmorilloniteBasic Red R1 C.I. 45160 conjugate, Montmorillonite C.I. Basic Black 2conjugate, Hectorite Basic Blue B7 C.I. 42595 conjugate, Hectorite BasicBlue B9 C.I. 52015 conjugate, Hectorite Basic Violet V3 C.I. 42555conjugate, Hectorite Basic Green G1 C.I. 42040 conjugate, HectoriteBasic Red R1 C.I. 45160 conjugate, Hectorite C.I. Basic Black 2conjugate, Saponite Basic Blue B7 C.I. 42595 conjugate, Saponite BasicBlue B9 C.I. 52015 conjugate, Saponite Basic Violet V3 C.I. 42555conjugate, Saponite Basic Green G1 C.I. 42040 conjugate, Saponite BasicRed R1 C.I. 45160 conjugate, Saponite C.I. Basic Black 2 conjugate andmixtures thereof.

Suitable pigments include pigments selected from the group consisting offlavanthrone, indanthrone, chlorinated indanthrone containing from 1 to4 chlorine atoms, pyranthrone, dichloropyranthrone,monobromodichloropyranthrone, dibromodichloropyranthrone,tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide,wherein the imide groups may be unsubstituted or substituted byC1-C3-alkyl or a phenyl or heterocyclic radical, and wherein the phenyland heterocyclic radicals may additionally carry substituents which donot confer solubility in water, anthrapyrimidinecarboxylic acid amides,violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyaninewhich may contain up to 2 chlorine atoms per molecule, polychloro-copperphthalocyanine or polybromochloro-copper phthalocyanine containing up to14 bromine atoms per molecule and mixtures thereof.

In one aspect, suitable pigments include pigments selected from thegroup consisting of Ultramarine Blue (C.I. Pigment Blue 29), UltramarineViolet (C.I. Pigment Violet 15) and mixtures thereof.

The aforementioned fabric hueing agents can be used in combination (anymixture of fabric hueing agents can be used). Suitable hueing agents aredescribed in more detail in U.S. Pat. No. 7,208,459. Preferred levels ofdye in compositions of the invention are 0.00001 to 0.5 wt %, or 0.0001to 0.25 wt %. The concentration of dyes preferred in water for thetreatment and/or cleaning step is from 1 ppb to 5 ppm, 10 ppb to 5 ppmor 20 ppb to 5 ppm. In preferred compositions, the concentration ofsurfactant will be from 0.2 to 3 g/l.

Encapsulates—

The composition may comprise an encapsulate. In one aspect, anencapsulate comprising a core, a shell having an inner and outersurface, said shell encapsulating said core.

In one aspect of said encapsulate, said core may comprise a materialselected from the group consisting of perfumes; brighteners; dyes;insect repellants; silicones; waxes; flavors; vitamins; fabric softeningagents; skin care agents in one aspect, paraffins; enzymes;anti-bacterial agents; bleaches; sensates; and mixtures thereof; andsaid shell may comprise a material selected from the group consisting ofpolyethylenes; polyamides; polyvinylalcohols, optionally containingother co-monomers; polystyrenes; polyisoprenes; polycarbonates;polyesters; polyacrylates; aminoplasts, in one aspect said aminoplastmay comprise a polyureas, polyurethane, and/or polyureaurethane, in oneaspect said polyurea may comprise polyoxymethyleneurea and/or melamineformaldehyde; polyolefins; polysaccharides, in one aspect saidpolysaccharide may comprise alginate and/or chitosan; gelatin; shellac;epoxy resins; vinyl polymers; water insoluble inorganics; silicone; andmixtures thereof.

In one aspect of said encapsulate, said core may comprise perfume.

In one aspect of said encapsulate, said shell may comprise melamineformaldehyde and/or cross linked melamine formaldehyde.

In a one aspect, suitable encapsulates may comprise a core material anda shell, said shell at least partially surrounding said core material,is disclosed. At least 75%, 85% or 90% of said encapsulates may have afracture strength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to3.5 MPa, or from 0.7 to 3 MPa; and a benefit agent leakage of from 0 to30%, from 0 to 20%, or from 0 to 5%.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle size from 1 to 80 microns, from 5 to 60 microns, from 10 to 50microns, or from 15 to 40 microns.

In one aspect, at least 75%, 85% or 90% of said encapsulates may have aparticle wall thickness from 30 to 250 nm, from 80 to 180 nm, or from100 to 160 nm.

In one aspect, said encapsulates' core material may comprise a materialselected from the group consisting of a perfume raw material and/oroptionally a material selected from the group consisting of vegetableoil, including neat and/or blended vegetable oils including castor oil,coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil,palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconutoil, palm kernel oil, castor oil, lemon oil and mixtures thereof; estersof vegetable oils, esters, including dibutyl adipate, dibutyl phthalate,butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate,trioctyl phosphate and mixtures thereof; straight or branched chainhydrocarbons, including those straight or branched chain hydrocarbonshaving a boiling point of greater than about 80° C.; partiallyhydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, includingmonoisopropylbiphenyl, alkylated naphthalene, includingdipropylnaphthalene, petroleum spirits, including kerosene, mineral oiland mixtures thereof; aromatic solvents, including benzene, toluene andmixtures thereof; silicone oils; and mixtures thereof.

In one aspect, said encapsulates' wall material may comprise a suitableresin including the reaction product of an aldehyde and an amine,suitable aldehydes include, formaldehyde. Suitable amines includemelamine, urea, benzoguanamine, glycoluril, and mixtures thereof.Suitable melamines include methylol melamine, methylated methylolmelamine, imino melamine and mixtures thereof. Suitable ureas includedimethylol urea, methylated dimethylol urea, urea-resorcinol, andmixtures thereof.

In one aspect, suitable formaldehyde scavengers may be employed with theencapsulates e.g. in a capsule slurry and/or added to a compositionbefore, during or after the encapsulates are added to such composition.Suitable capsules may be made by the following teaching ofUS2008/0305982; and/or US2009/0247449.

In a preferred aspect the composition can also comprise a depositionaid, preferably consisting of the group comprising cationic or nonionicpolymers. Suitable polymers include cationic starches, cationichydroxyethylcellulose, polyvinyl-formaldehyde, locust bean gum, mannans,xyloglucans, tamarind gum, polyethylene terephthalate and polymerscontaining dimethylaminoethyl methacrylate, optionally with one ormonomers selected from the group comprising acrylic acid and acrylamide.

Perfumes—In one aspect the composition comprises a perfume thatcomprises one or more perfume raw materials selected from the groupconsisting of 1,1′-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid,diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol,monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol;2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol;oxacyclohexadecan-2-one; alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; beta-methylbenzenepropanal; beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde;2-methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl-2(3H)furanone;(2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal;undecanal; 2-ethyl-alpha, alpha-dimethylbenzenepropanal; decanal; alpha,alpha-dimethylbenzeneethanol acetate; 2-(phenylmethylene)octanal;2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoicacid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one;2-pentylcyclopentanone; 3-oxo-2-pentyl cyclopentaneacetic acid, methylester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde;2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone;(3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;benzeneethanol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde;10-undecenal; propanoic acid, phenylmethyl ester;beta-methylbenzenepentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene;alpha, alpha-dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid,phenylmethyl ester; cyclohexanepropanoic acid, 2-propenyl ester;hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene;1,5-dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-buten-2-ol; 2-[[[2,4(or3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methylester; 8-cyclohexadecen-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol;2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-Octadien-1-ol;2-hydroxy-Benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenenitrile;4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; Acetic acid,(2-methylbutoxy)-, 2-propenyl ester; Benzoic acid,2-hydroxy-3-methylbutyl ester; 2-Buten-1-one,1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; Cyclopentanecarboxylicacid, 2-hexyl-3-oxo-, methyl ester; Benzenepropanal,4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde,3-(4-hydroxy-4-methylpentyl)-; Ethanone,1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-,[3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]-; Undecanal,2-methyl-2H-Pyran-2-one, 6-butyltetrahydro-; Benzenepropanal,4-(1,1-dimethylethyl)-.alpha.-methyl-; 2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester; Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-; 2(3H)-Furanone, 5-hexyldihydro-;Oxiranecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzenepentanol,.gamma.-methyl-; 3-Octanol, 3,7-dimethyl-;3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; Terpineolacetate; 2-methyl-6-methylene-7-Octen-2-ol, dihydro derivative;3a,4,5,6,7,7a-hexahydro-4,7-Methano-1H-inden-6-ol propanoate;3-methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate;2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-ethanone;2-hydroxy-benzoic acid, methyl ester; 2-hydroxy-benzoic acid, hexylester; 2-phenoxy-ethanol; 2-hydroxy-benzoic acid, pentyl ester;2,3-heptanedione; 2-hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; damascone(alpha, beta, gamma or delta or mixtures thereof),4,7-Methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate;9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone,1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-;3-Cyclohexene-1-carboxaldehyde, 3,5-dimethyl-;3-Cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-Octadien-3-ol,3,7-dimethyl-; 1,6-Octadien-3-ol, 3,7-dimethyl-, acetate; Lilial(p-t-Bucinal), and Cyclopentanone,2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and1-methyl-4-(1-methylethenyl)cyclohexene and mixtures thereof.

In one aspect the composition may comprise an encapsulated perfumeparticle comprising either a water-soluble hydroxylic compound ormelamine-formaldehyde or modified polyvinyl alcohol. In one aspect theencapsulate comprises (a) an at least partially water-soluble solidmatrix comprising one or more water-soluble hydroxylic compounds,preferably starch; and (b) a perfume oil encapsulated by the solidmatrix.

In a further aspect the perfume may be pre-complexed with a polyamine,preferably a polyethylenimine so as to form a Schiff base.

Polymers—

The composition may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

The composition may comprise one or more amphiphilic cleaning polymerssuch as the compound having the following general structure:bis((C₂H₅O)(C₂H₄O)n)(CH₃)—N—C_(x)H_(2x)—N⁺—(CH₃)-bis((C₂H₅O)(C₂H₄O)n),wherein n=from 20 to 30, and x=from 3 to 8, or sulphated or sulphonatedvariants thereof.

The composition may comprise amphiphilic alkoxylated grease cleaningpolymers which have balanced hydrophilic and hydrophobic properties suchthat they remove grease particles from fabrics and surfaces. Specificaspects of the amphiphilic alkoxylated grease cleaning polymers of thepresent invention comprise a core structure and a plurality ofalkoxylate groups attached to that core structure. These may comprisealkoxylated polyalkylenimines, preferably having an inner polyethyleneoxide block and an outer polypropylene oxide block.

Alkoxylated polycarboxylates such as those prepared from polyacrylatesare useful herein to provide additional grease removal performance. Suchmaterials are described in WO91/08281 and PCT90/01815. Chemically, thesematerials comprise polyacrylates having one ethoxy side-chain per every7-8 acrylate units. The side-chains are of the formula—(CH₂CH₂O)_(m)(CH₂)_(n)CH₃ wherein m is 2-3 and n is 6-12. Theside-chains are ester-linked to the polyacrylate “backbone” to provide a“comb” polymer type structure. The molecular weight can vary, but istypically in the range of 2000 to 50,000. Such alkoxylatedpolycarboxylates can comprise from 0.05 wt % to 10 wt % of thecompositions herein.

The isoprenoid-derived surfactants of the present invention, and theirmixtures with other cosurfactants and other adjunct ingredients, areparticularly suited to be used with an amphilic graft co-polymer,preferably the amphilic graft co-polymer comprises (i) polyethyeleneglycol backbone; and (ii) and at least one pendant moiety selected frompolyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferredamphilic graft co-polymer is Sokalan HP22, supplied from BASF. Suitablepolymers include random graft copolymers, preferably a polyvinyl acetategrafted polyethylene oxide copolymer having a polyethylene oxidebackbone and multiple polyvinyl acetate side chains. The molecularweight of the polyethylene oxide backbone is preferably 6000 and theweight ratio of the polyethylene oxide to polyvinyl acetate is 40 to 60and no more than 1 grafting point per 50 ethylene oxide units.

Carboxylate Polymer—

The composition of the present invention may also include one or morecarboxylate polymers such as a maleate/acrylate random copolymer orpolyacrylate homopolymer. In one aspect, the carboxylate polymer is apolyacrylate homopolymer having a molecular weight of from 4,000 to9,000 Da, or from 6,000 to 9,000 Da.

Soil Release Polymer—

The composition of the present invention may also include one or moresoil release polymers having a structure as defined by one of thefollowing structures (I), (II) or (Ill):—[(OCHR¹—CHR²)_(a)—O—OC—Ar—CO-]_(d)  (I)—[(OCHR³—CHR⁴)_(b)—O—OC-sAr-CO-]_(e)  (II)—[(OCHR⁵—CHR⁶)_(c)—OR⁷]_(f)  (III)wherein:a, b and c are from 1 to 200;d, e and f are from 1 to 50;Ar is a 1,4-substituted phenylene;sAr is 1,3-substituted phenylene substituted in position 5 with SO₃Me;Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, mono-, di-, tri-, ortetraalkylammonium wherein the alkyl groups are C₁-C₁₈ alkyl or C₂-C₁₀hydroxyalkyl, or mixtures thereof;R¹, R², R³, R⁴, R⁵ and R⁶ are independently selected from H or C₁-C₈ n-or iso-alkyl; andR⁷ is a linear or branched C₁-C₁₈ alkyl, or a linear or branched C₂-C₃₀alkenyl, or a cycloalkyl group with 5 to 9 carbon atoms, or a C₈-C₃₀aryl group, or a C₆-C₃₀ arylalkyl group.

Suitable soil release polymers are polyester soil release polymers suchas Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6 suppliedby Rhodia. Other suitable soil release polymers include Texcarepolymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240,SRN300 and SRN325 supplied by Clariant. Other suitable soil releasepolymers are Marloquest polymers, such as Marloquest SL supplied bySasol.

Cellulosic Polymer—

The composition of the present invention may also include one or morecellulosic polymers including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl carboxyalkylcellulose. In one aspect, the cellulosic polymers are selected from thegroup comprising carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixuresthereof. In one aspect, the carboxymethyl cellulose has a degree ofcarboxymethyl substitution from 0.5 to 0.9 and a molecular weight from100,000 to 300,000 Da.

Enzymes—

The composition may comprise one or more enzymes which provide cleaningperformance and/or fabric care benefits. Examples of suitable enzymesinclude, but are not limited to, hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, mannanases, pectate lyases, keratinases, reductases,oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, malanases, R-glucanases, arabinosidases,hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, ormixtures thereof. A typical combination is an enzyme cocktail that maycomprise e.g. a protease and lipase in conjunction with amylase. Whenpresent in a composition, the aforementioned additional enzymes may bepresent at levels from 0.00001 to 2 wt %, from 0.0001 to 1 wt % or from0.001 to 0.5 wt % enzyme protein by weight of the composition.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

In one aspect preferred enzymes would include a cellulase. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP0495257, EP0531372, WO96/11262, WO96/29397, WO98/08940.Other examples are cellulase variants such as those described inWO94/07998, EP0531315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254,WO95/24471, WO98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™Celluclean, Carezyme Premium, Whitezyme (Novozymes A/S), Clazinase™, andPuradax HA™ (Genencor International Inc.), and KAC-500(B)™ (KaoCorporation).

In one aspect preferred enzymes would include a protease. Suitableproteases include those of bacterial, fungal, plant, viral or animalorigin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered mutants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteasesprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO009/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO005/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens. Examples of useful proteases are thevariants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116,WO99/011768, WO01/44452, WO003/006602, WO04/03186, WO04/041979,WO07/006305, WO11/036263, WO11/036264, especially the variants withsubstitutions in one or more of the following positions: 3, 4, 9, 15,27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205,206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using theBPN′ numbering. More preferred the subtilase variants may comprise themutations: S3T, V41, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E,A98S, S99G,D,A, S99AD, S101G,M,R S103A, V1041,Y,N, S106A, G118V,R,H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E,V199M, V2051, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K,T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Blaze®; Duralase™, Durazym™, Relase®,Savinase®, Primase®, Polarzyme®, Kannase®, Liquanase®, Ovozyme®,Coronase®, Neutrase®, Everlase® and Esperase® all could be sold asUltra® or Evity® (Novozymes A/S), those sold under the tradenameMaxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®,Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase®(Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown inFIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) andKAP (Bacillus alkalophilus subtilisin) from Kao.

In one aspect, preferred enzymes would include an amylase. Suitableamylases may be an alpha-amylase or a glucoamylase and may be ofbacterial orfungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of Bacilluslicheniformis, described in more detail in GB1296839.

Suitable amylases include amylases having SEQ ID NO: 3 in WO95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO94/02597, WO94/18314, WO97/43424 and SEQ IDNO: 4 of WO99/019467, such as variants with substitutions in one or moreof the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156,178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264,304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 inWO02/010355 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:M197T; H156Y+A181T+N190F+A209V+Q264S; orG48A+T491+G107A+H156Y+A181T+N190F+1201 F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which can be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. Morepreferred variants are those having a deletion in positions 181 and 182or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions183 and 184 and a substitution in one or more of positions 140, 195,206, 243, 260, 304 and 476.

Other amylases which can be used are amylases having SEQ ID NO: 2 ofWO008/153815, SEQ ID NO: 10 in WO01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO008/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO01/66712. Preferred variants of SEQ IDNO: 10 in WO01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 ofWO009/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T1651, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or 0183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:N128C+K178L+T182G+Y305R+G475K;N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;S125A+N128C+K178L+T182G+Y305R+G475K; orS125A+N128C+T131I+T1651+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, TermamylUltra™, Fungamyl™, Ban™, Stainzyme™, Stainzyme Plus™, Amplify®,Supramyl™, Natalase™ Liquozyme X, BAN™, Resillience and Everest (fromNovozymes A/S), KEMZYM® AT 9000 Biozym Biotech Trading GmbH Wehlistrasse27b A-1200 Wien Austria, and Rapidase™ Purastar™/Effectenz™, Powerase,Preferenz S100, Preferenx S110, ENZYSIZE®, OPTISIZE HT PLUS®, andPURASTAR OXAM® (Danisco/DuPont) and KAM® (Kao).

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412, WO13/033318), Geobacillusstearothermophilus lipase (WO11/084417), lipase from Bacillus subtilis(WO11/084599), and lipase from Streptomyces griseus (WO11/150157) and S.pristinaespiralis (WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include Lipolase™, Lipex™, LipexEvity™, Lipolex™, Lipoclean™ and Lipex Evity 100 L (Novozymes A/S),Lumafast (originally from DuPont) and Lipomax™ (originally fromGist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO 10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

In one aspect, other preferred enzymes include microbial-derivedendoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4),including a bacterial polypeptide endogenous to a member of the genusBacillus which has a sequence of at least 90%, 94%, 97% or 99% identityto the amino acid sequence SEQ ID NO:2 in U.S. Pat. No. 7,141,403 andmixtures thereof. Suitable endoglucanases are sold under the tradenamesCelluclean® and Whitezyme® (Novozymes).

Other preferred enzymes include pectate lyases sold under the tradenamesPectawash®, Pectaway®, Xpect® and mannanases sold under the tradenamesMannaway® (Novozymes), and Purabrite® (Danisco/DuPont).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all or multiple of these enzymes. Adetergent additive of the invention, i.e., a separate additive or acombined additive, can be formulated, for example, as granulate, liquid,slurry, bar etc. Preferred detergent additive formulations aregranulates, in particular non-dusting granulates, liquids, in particularstabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Protected enzymes may beprepared according to the method disclosed in EP238216.

Dye Transfer Inhibiting Agents—

The compositions of the present invention may also include one or moredye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a composition, the dye transferinhibiting agents may be present at levels from 0.0001 to 10 wt %, from0.01 to 5 wt % or from 0.1 to 3 wt %.

Brighteners—

The compositions of the present invention can also contain additionalcomponents that may tint articles being cleaned, such as fluorescentbrighteners.

The composition may comprise C.I. fluorescent brightener 260 inalpha-crystalline form having the following structure:

In one aspect, the brightener is a cold water soluble brightener, suchas the C.I. fluorescent brightener 260 in alpha-crystalline form. In oneaspect the brightener is predominantly in alpha-crystalline form, whichmeans that typically at least 50 wt %, at least 75 wt %, at least 90 wt%, at least 99 wt %, or even substantially all, of the C.I. fluorescentbrightener 260 is in alpha-crystalline form.

The brightener is typically in micronized particulate form, having aweight average primary particle size of from 3 to 30 micrometers, from 3micrometers to 20 micrometers, or from 3 to 10 micrometers.

The composition may comprise C.I. fluorescent brightener 260 inbeta-crystalline form, and the weight ratio of: (i) C.I. fluorescentbrightener 260 in alpha-crystalline form, to (ii) C.I. fluorescentbrightener 260 in beta-crystalline form may be at least 0.1, or at least0.6. BE680847 relates to a process for making C.I fluorescent brightener260 in alpha-crystalline form.

Commercial optical brighteners which may be useful in the presentinvention can be classified into subgroups, which include, but are notnecessarily limited to, derivatives of stilbene, pyrazoline, coumarin,carboxylic acid, methinecyanines, dibenzothiophene-5,5-dioxide, azoles,5- and 6-membered-ring heterocycles, and other miscellaneous agents.Examples of such brighteners are disclosed in “The Production andApplication of Fluorescent Brightening Agents”, M. Zahradnik, Publishedby John Wiley & Sons, New York (1982). Specific nonlimiting examples ofoptical brighteners which are useful in the present compositions arethose identified in U.S. Pat. Nos. 4,790,856 and 3,646,015.

A further suitable brightener has the structure below:

Suitable fluorescent brightener levels include lower levels of from 0.01wt %, from 0.05 wt %, from 0.1 wt % or from 0.2 wt % to upper levels of0.5 wt % or 0.75 wt %.

In one aspect the brightener may be loaded onto a clay to form aparticle. Silicate salts—The compositions of the present invention canalso contain silicate salts, such as sodium or potassium silicate. Thecomposition may comprise of from 0 wt % to less than 10 wt % silicatesalt, to 9 wt %, or to 8 wt %, or to 7 wt %, or to 6 wt %, or to 5 wt %,or to 4 wt %, or to 3 wt %, or even to 2 wt %, and from above 0 wt %, orfrom 0.5 wt %, or from 1 wt % silicate salt. A suitable silicate salt issodium silicate.

Dispersants—

The compositions of the present invention can also contain dispersants.Suitable water-soluble organic materials include the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

Enzyme Stabilizers—

Enzymes for use in compositions can be stabilized by various techniques.The enzymes employed herein can be stabilized by the presence ofwater-soluble sources of calcium and/or magnesium ions. Examples ofconventional stabilizing agents are, e.g. a polyol such as propyleneglycol or glycerol, a sugar or sugar alcohols, and lactic acid. Thedetergent composition may include a protease inhibitor, which is areversible inhibitor of protease activity, e.g., serine proteaseactivity. Preferably, the protease inhibitor is a (reversible)subtilisin protease inhibitor. In particular, the protease inhibitor maybe a peptide aldehyde, boric acid, or a boronic acid; or a derivative ofany of these.

The protease inhibitor may have an inhibition constant to a serineprotease, Ki (mol/L) of from 1E-12 to 1E-03; more preferred from 1E-11to 1E-04; even more preferred from 1E-10 to 1E-05; even more preferredfrom 1E-10 to 1E-06; and most preferred from 1E-09 to 1E-07.

The protease inhibitor may be boronic acid or a derivative thereof;preferably, phenylboronic acid or a derivative thereof.

In one aspect, the phenyl boronic acid derivative is of the followingformula:

wherein R is selected from the group consisting of hydrogen, hydroxy,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkenyl and substitutedC₁-C₆ alkenyl. Preferably, R is hydrogen, CH₃, CH₃CH₂ or CH₃CH₂CH₂.

In a preferred aspect, the protease inhibitor (phenyl boronic acidderivative) is 4-formyl-phenyl-boronic acid (4-FPBA).

In another particular aspect, the protease inhibitor is selected fromthe group consisting of:

thiophene-2 boronic acid, thiophene-3 boronic acid, acetamidophenylboronic acid, benzofuran-2 boronic acid, naphtalene-1 boronic acid,naphtalene-2 boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thianthrene boronicacid, 4-dibenzofuran boronic acid, 5-methylthiophene-2 boronic, acid,thionaphtrene boronic acid, furan-2 boronic acid, furan-3 boronic acid,4,4 biphenyl-diborinic acid, 6-hydroxy-2-naphtalene, 4-(methylthio)phenyl boronic acid, 4 (trimethyl-silyl)phenyl boronic acid,3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphtylboronic acid, 5-bromothiphene boronic acid, 5-chlorothiophene boronicacid, dimethylthiophene boronic acid, 2-bromophenyl boronic acid,3-chlorophenyl boronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thianthrene boronic acid, di-benzothiophene boronicacid, 4-carboxyphenyl boronic acid, 9-anthryl boronic acid, 3,5dichlorophenyl boronic, acid, diphenyl boronic acidanhydride,o-chlorophenyl boronic acid, p-chlorophenyl boronic acid, m-bromophenylboronic acid, p-bromophenyl boronic acid, p-flourophenyl boronic acid,p-tolyl boronic acid, o-tolyl boronic acid, octyl boronic acid, 1,3,5trimethylphenyl boronic acid, 3-chloro-4-flourophenyl boronic acid,3-aminophenyl boronic acid, 3,5-bis-(triflouromethyl) phenyl boronicacid, 2,4 dichlorophenyl boronic acid, 4-methoxyphenyl boronic acid.

Further boronic acid derivatives suitable as protease inhibitors in thedetergent composition are described in U.S. Pat. Nos. 4,963,655,5,159,060, WO95/12655, WO95/29223, WO92/19707, WO94/04653, WO94/04654,U.S. Pat. Nos. 5,442,100, 5,488,157 and 5,472,628.

The protease inhibitor may also be a peptide aldehyde having the formulaX—B¹—B—H, wherein the groups have the following meaning:

a) H is hydrogen;

b) B⁰ is a single amino acid residue with L- or D-configuration and withthe formula: NH—CHR′—CO;

c) B¹ is a single amino acid residue; and

d) X consists of one or more amino acid residues (preferably one ortwo), optionally comprising an N-terminal protection group.

NH—CHR′—CO (B⁰) is an L or D-amino acid residue, where R′ may be analiphatic or aromatic side chain, e.g., aralkyl, such as benzyl, whereR′ may be optionally substituted. More particularly, the B⁰ residue maybe bulky, neutral, polar, hydrophobic and/or aromatic. Examples are theD- or L-form of Tyr (p-tyrosine), m-tyrosine,3,4-dihydroxyphenylalanine, Phe, Val, Met, norvaline (Nva), Leu, Ile ornorleucine (Nle).

In the above formula, X—B¹—B—H, the B¹ residue may particularly besmall, aliphatic, hydrophobic and/or neutral. Examples are alanine(Ala), cysteine (Cys), glycine (Gly), proline (Pro), serine (Ser),threonine (Thr), valine (Val), norvaline (Nva) and norleucine (Nle),particularly alanine, glycine, or valine.

X may in particular be one or two amino acid residues with an optionalN-terminal protection group (i.e. the compound is a tri- or tetrapeptidealdehyde with or without a protection group). Thus, X may be B², B³—B²,Z—B², or Z—B³—B² where B³ and B² each represents one amino acid residue,and Z is an N-terminal protection group. The B² residue may inparticular be small, aliphatic and/or neutral, e.g., Ala, Gly, Thr, Arg,Leu, Phe or Val. The B³ residue may in particular be bulky, hydrophobic,neutral and/or aromatic, e.g., Phe, Tyr, Trp, Phenylglycine, Leu, Val,Nva, Nle or lie.

The N-terminal protection group Z (if present) may be selected fromformyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxy carbonyl,methoxysuccinyl, aromatic and aliphatic urethane protecting groups,benzyloxycarbonyl (Cbz), t-butyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) orp-methoxyphenyl (PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac);methyl carbamate or a methylamino carbonyl/methyl urea group. In thecase of a tripeptide aldehyde with a protection group (i.e. X═Z—B²), Zis preferably a small aliphatic group, e.g., formyl, acetyl,fluoromethoxy carbonyl, t-butyloxycarbonyl, methoxycarbonyl (Moc);methoxyacetyl (Mac); methyl carbamate or a Methylamino carbonyl/methylurea group. In the case of a tripeptide aldehyde with a protection group(i.e. X═Z—B³—B²), Z is preferably a bulky aromatic group such asbenzoyl, benzyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl (Bn),p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP).

Suitable peptide aldehydes are described in WO94/04651, WO95/25791,WO98/13458, WO98/13459, WO98/13460, WO98/13461, WO98/13461, WO98/13462,WO07/141736, WO07/145963, WO09/118375, WO10/055052 and WO11/036153. Moreparticularly, the peptide aldehyde may be Cbz-RAY-H, Ac-GAY-H,Cbz-GAY-H, Cbz-GAL-H, Cbz-VAL-H, Cbz-GAF-H, Cbz-GAV-H, Cbz-GGY-H,Cbz-GGF-H, Cbz-RVY-H, Cbz-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H,Ac-FGAL-H, Ac-FGAF-H, Ac-FGVY-H, Ac-FGAM-H, Ac-WLVY-H, MeO—CO-VAL-H,MeNCO-VAL-H, MeO—CO-FGAL-H, MeO—CO-FGAF-H, MeSO₂-FGAL-H, MeSO₂-VAL-H,PhCH₂O(OH)(O)P-VAL-H, EtSO₂-FGAL-H, PhCH₂SO₂-VAL-H,PhCH₂O(OH)(O)P-LAL-H, PhCH₂O(OH)(O)P-FAL-H, or MeO(OH)(O)P-LGAL-H. Here,Cbz is benzyloxycarbonyl, Me is methyl, Et is ethyl, Ac is acetyl, H ishydrogen, and the other letters represent amino acid residues denoted bystandard single letter notification (e.g., F=Phe, Y=Tyr, L=Leu).

Alternatively, the peptide aldehyde may have the formula as described inWO11/036153:P—O-(A_(i)-X′)_(n)-A_(n+1)-Q

wherein Q is hydrogen, CH₃, CX″₃, CHX″₂, or CH₂X″, wherein X″ is ahalogen atom;

wherein one X′ is the “double N-capping group” CO, CO—CO, CS, CS—CS orCS—CO, most preferred urido (CO), and the other X′ are nothing,

wherein n=1-10, preferably 2-5, most preferably 2,

wherein each of A_(i) and A_(n+1) is an amino acid residue having thestructure:

—NH—CR″—CO— for a residue to the right of X′=—CO—, or

—CO—CR″—NH— for a residue to the left of X′=—CO—

wherein R″ is H— or an optionally substituted alkyl or alkylaryl groupwhich may optionally include a hetero atom and may optionally be linkedto the N atom, and

wherein P is hydrogen or any C-terminal protection group.

Examples of such peptide aldehydes include α-MAPI, β-MAPI, F-urea-RVY-H,F-urea-GGY-H, F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H,F-urea-GA-Nle-H, Y-urea-RVY-H, Y-urea-GAY-H, F-CS-RVF-H, F-CS-RVY-H,F-CS-GAY-H, Antipain, GE20372A, GE20372B, Chymostatin A, Chymostatin B,and Chymostatin C. Further examples of peptide aldehydes are disclosedin WO2010/055052 and WO2009/118375, WO94/04651, WO98/13459, WO98/13461,WO98/13462, WO07/145963, hereby incorporated by reference.

Alternatively, to a peptide aldehyde, the protease inhibitor may be ahydrosulfite adduct having the formula X—B¹—NH—CHR—CHOH—SO₃M, wherein X,B¹ and R are defined as above, and M is H or an alkali metal, preferablyNa or K as described WO13/004636.

The peptide aldehyde may be converted into a water-soluble hydrosulfiteadduct by reaction with sodium bisulfite, as described in textbooks,e.g., March, J. Advanced Organic Chemistry, fourth edition,Wiley-lnterscience, US 1992, p 895.

An aqueous solution of the bisulfite adduct may be prepared by reactingthe corresponding peptide aldehyde with an aqueous solution of sodiumbisulfite (sodium hydrogen sulfite, NaHSO₃); potassium bisulfite (KHSO₃)by known methods, e.g., as described in WO98/47523; U.S. Pat. No.6,500,802; U.S. Pat. No. 5,436,229; J. Am. Chem. Soc. (1978) 100, 1228;Org. Synth., Coil. vol. 7: 361.

The molar ratio of the above-mentioned peptide aldehydes (orhydrosulfite adducts) to the protease may be at least 1:1 or 1.5:1, andit may be less than 1000:1, more preferred less than 500:1, even morepreferred from 100:1 to 2:1 or from 20:1 to 2:1, or most preferred, themolar ratio is from 10:1 to 2:1.

Formate salts (e.g., sodium formate) and formic acid have also showngood effects as inhibitor of protease activity. Formate can be usedsynergistically with the above-mentioned protease inhibitors, as shownin WO 13/004635. The formate salts may be present in the detergentcomposition in an amount of at least 0.1% w/w or 0.5% w/w, e.g., atleast 1.0%, at least 1.2% or at least 1.5%. The amount of the salt istypically below 5% w/w, below 4% or below 3%.

In an aspect, the protease is a metalloprotease and the inhibitor is ametalloprotease inhibitor, e.g., a protein hydrolysate based inhibitor(e.g., as described in WO08/134343).

Solvents—

Suitable solvents include water and other solvents such as lipophilicfluids. Examples of suitable lipophilic fluids include siloxanes, othersilicones, hydrocarbons, glycol ethers, glycerine derivatives such asglycerine ethers, perfluorinated amines, perfluorinated andhydrofluoroether solvents, low-volatility nonfluorinated organicsolvents, diol solvents, other environmentally-friendly solvents andmixtures thereof.

Structurant/Thickeners—

Structured liquids can either be internally structured, whereby thestructure is formed by primary ingredients (e.g. surfactant material)and/or externally structured by providing a three-dimensional matrixstructure using secondary ingredients (e.g. polymers, clay and/orsilicate material). The composition may comprise a structurant, from0.01 to 5 wt %, or from 0.1 to 2.0 wt %. The structurant is typicallyselected from the group consisting of diglycerides and triglycerides,ethylene glycol distearate, microcrystalline cellulose, cellulose-basedmaterials, microfiber cellulose, hydrophobically modifiedalkali-swellable emulsions such as Polygel W30 (3VSigma), biopolymers,xanthan gum, gellan gum, and mixtures thereof. A suitable structurantincludes hydrogenated castor oil, and non-ethoxylated derivativesthereof. A suitable structurant is disclosed in U.S. Pat. No. 6,855,680.Such structurants have a thread-like structuring system having a rangeof aspect ratios. Other suitable structurants and the processes formaking them are described in WO10/034736.

Conditioning Agents—

The composition of the present invention may include a high meltingpoint fatty compound. The high melting point fatty compound usefulherein has a melting point of 25° C. or higher, and is selected from thegroup consisting of fatty alcohols, fatty acids, fatty alcoholderivatives, fatty acid derivatives, and mixtures thereof. Suchcompounds of low melting point are not intended to be included in thissection. Non-limiting examples of the high melting point compounds arefound in International Cosmetic Ingredient Dictionary, Fifth Edition,1993, and CTFA Cosmetic Ingredient Handbook, Second Edition, 1992.

The high melting point fatty compound is included in the composition ata level of from 0.1 to 40 wt %, from 1 to 30 wt %, from 1.5 to 16 wt %,from 1.5 to 8 wt % in view of providing improved conditioning benefitssuch as slippery feel during the application to wet hair, softness andmoisturized feel on dry hair.

The compositions of the present invention may contain a cationicpolymer. Concentrations of the cationic polymer in the compositiontypically range from 0.05 to 3 wt %, from 0.075 to 2.0 wt %, or from 0.1to 1.0 wt %. Suitable cationic polymers will have cationic chargedensities of at least 0.5 meq/gm, at least 0.9 meq/gm, at least 1.2meq/gm, at least 1.5 meq/gm, or less than 7 meq/gm, and less than 5meq/gm, at the pH of intended use of the composition, which pH willgenerally range from pH3 to pH9, or between pH4 and pH8. Herein,“cationic charge density” of a polymer refers to the ratio of the numberof positive charges on the polymer to the molecular weight of thepolymer. The average molecular weight of such suitable cationic polymerswill generally be between 10,000 and 10 million, between 50,000 and 5million, or between 100,000 and 3 million.

Suitable cationic polymers for use in the compositions of the presentinvention contain cationic nitrogen-containing moieties such asquaternary ammonium or cationic protonated amino moieties. Any anioniccounterions can be used in association with the cationic polymers solong as the polymers remain soluble in water, in the composition, or ina coacervate phase of the composition, and so long as the counterionsare physically and chemically compatible with the essential componentsof the composition or do not otherwise unduly impair compositionperformance, stability or aesthetics. Nonlimiting examples of suchcounterions include halides (e.g., chloride, fluoride, bromide, iodide),sulfate and methylsulfate.

Nonlimiting examples of such polymers are described in the CTFA CosmeticIngredient Dictionary, 3rd edition, edited by Estrin, Crosley, andHaynes, (The Cosmetic, Toiletry, and Fragrance Association, Inc.,Washington, D.C. (1982)).

Other suitable cationic polymers for use in the composition includepolysaccharide polymers, cationic guar gum derivatives, quaternarynitrogen-containing cellulose ethers, synthetic polymers, copolymers ofetherified cellulose, guar and starch. When used, the cationic polymersherein are either soluble in the composition or are soluble in a complexcoacervate phase in the composition formed by the cationic polymer andthe anionic, amphoteric and/or zwitterionic surfactant componentdescribed hereinbefore. Complex coacervates of the cationic polymer canalso be formed with other charged materials in the composition. Suitablecationic polymers are described in U.S. Pat. Nos. 3,962,418; 3,958,581;and US2007/0207109.

The composition of the present invention may include a nonionic polymeras a conditioning agent. Polyalkylene glycols having a molecular weightof more than 1000 are useful herein. Useful are those having thefollowing general formula:

wherein R⁹⁵ is selected from the group consisting of H, methyl, andmixtures thereof. Conditioning agents, and in particular silicones, maybe included in the composition. The conditioning agents useful in thecompositions of the present invention typically comprise a waterinsoluble, water dispersible, non-volatile, liquid that formsemulsified, liquid particles. Suitable conditioning agents for use inthe composition are those conditioning agents characterized generally assilicones (e.g., silicone oils, cationic silicones, silicone gums, highrefractive silicones, and silicone resins), organic conditioning oils(e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinationsthereof, or those conditioning agents which otherwise form liquid,dispersed particles in the aqueous surfactant matrix herein. Suchconditioning agents should be physically and chemically compatible withthe essential components of the composition, and should not otherwiseunduly impair composition stability, aesthetics or performance.

The concentration of the conditioning agent in the composition should besufficient to provide the desired conditioning benefits. Suchconcentration can vary with the conditioning agent, the conditioningperformance desired, the average size of the conditioning agentparticles, the type and concentration of other components, and otherlike factors.

The concentration of the silicone conditioning agent typically rangesfrom 0.01 to 10 wt %. Non-limiting examples of suitable siliconeconditioning agents, and optional suspending agents for the silicone,are described in U.S. Reissue Pat. No. 34,584; U.S. Pat. Nos. 5,104,646;5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837; 6,607,717;6,482,969; 5,807,956; 5,981,681; 6,207,782; 7,465,439; 7,041,767;7,217,777; US2007/0286837A1; US2005/0048549A1; US2007/0041929A1;GB849433; DE10036533, which are all incorporated herein by reference;Chemistry and Technology of Silicones, New York: Academic Press (1968);General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and inEncyclopedia of Polymer Science and Engineering, vol. 15, 2d ed., pp204-308, John Wiley & Sons, Inc. (1989).

The compositions of the present invention may also comprise from 0.05 to3 wt % of at least one organic conditioning oil as the conditioningagent, either alone or in combination with other conditioning agents,such as the silicones (described herein). Suitable conditioning oilsinclude hydrocarbon oils, polyolefins, and fatty esters. Also suitablefor use in the compositions herein are the conditioning agents describedin U.S. Pat. Nos. 5,674,478 and 5,750,122 or in U.S. Pat. Nos.4,529,586; 4,507,280; 4,663,158; 4,197,865; 4,217,914; 4,381,919; and4,422,853.

Hygiene and Malodour—

The compositions of the present invention may also comprise one or moreof zinc ricinoleate, thymol, quaternary ammonium salts such as Bardac®,polyethylenimines (such as Lupasol® from BASF) and zinc complexesthereof, silver and silver compounds, especially those designed toslowly release Ag′ or nano-silver dispersions.

Probiotics—

The compositions may comprise probiotics such as those described inWO09/043709.

Suds Boosters—

If high sudsing is desired, suds boosters such as the C₁₀-C₁₆alkanolamides or C₁₀-C₁₄ alkyl sulphates can be incorporated into thecompositions, typically at 1 to 10 wt % levels. The C₁₀-C₁₄ monoethanoland diethanol amides illustrate a typical class of such suds boosters.Use of such suds boosters with high sudsing adjunct surfactants such asthe amine oxides, betaines and sultaines noted above is alsoadvantageous. If desired, water-soluble magnesium and/or calcium saltssuch as MgCl₂, MgSO₄, CaCl₂, CaSO₄ and the like, can be added at levelsof, typically, 0.1 to 2 wt %, to provide additional suds and to enhancegrease removal performance.

Suds Suppressors—

Compounds for reducing or suppressing the formation of suds can beincorporated into the compositions of the present invention. Sudssuppression can be of particular importance in the so-called “highconcentration cleaning process” as described in U.S. Pat. Nos. 4,489,455and 4,489,574, and in front-loading-style washing machines. A widevariety of materials may be used as suds suppressors, and sudssuppressors are well known to those skilled in the art. See e.g. KirkOthmer Encyclopedia of Chemical Technology, Third Edition, Volume 7, p.430-447 (John Wiley & Sons, Inc., 1979). Examples of suds supressorsinclude monocarboxylic fatty acid and soluble salts therein, highmolecular weight hydrocarbons such as paraffin, fatty acid esters (e.g.,fatty acid triglycerides), fatty acid esters of monovalent alcohols,aliphatic C₁₈-C₄₀ ketones (e.g., stearone), N-alkylated amino triazines,waxy hydrocarbons preferably having a melting point below about 100° C.,silicone suds suppressors, and secondary alcohols. Suds supressors aredescribed in U.S. Pat. Nos. 2,954,347; 4,265,779; 4,265,779; 3,455,839;3,933,672; 4,652,392; 4,978,471; 4,983,316; 5,288,431; 4,639,489;4,749,740; 4,798,679; 4,075,118; EP89307851.9; EP150872; and DOS2,124,526.

For any detergent compositions to be used in automatic laundry washingmachines, suds should not form to the extent that they overflow thewashing machine. Suds suppressors, when utilized, are preferably presentin a “suds suppressing amount. By “suds suppressing amount” is meantthat the formulator of the composition can select an amount of this sudscontrolling agent that will sufficiently control the suds to result in alow-sudsing laundry detergent for use in automatic laundry washingmachines.

The compositions herein will generally comprise from 0 to 10 wt % ofsuds suppressor. When utilized as suds suppressors, monocarboxylic fattyacids, and salts therein, will be present typically in amounts up to 5wt %. Preferably, from 0.5 to 3 wt % of fatty monocarboxylate sudssuppressor is utilized. Silicone suds suppressors are typically utilizedin amounts up to 2.0 wt %, although higher amounts may be used.Monostearyl phosphate suds suppressors are generally utilized in amountsranging from 0.1 to 2 wt %. Hydrocarbon suds suppressors are typicallyutilized in amounts ranging from 0.01 to 5.0 wt %, although higherlevels can be used. The alcohol suds suppressors are typically used at0.2 to 3 wt %.

The compositions herein may have a cleaning activity over a broad rangeof pH. In certain aspects, the compositions have cleaning activity frompH4 to pH11.5. In other aspects, the compositions are active from pH6 topH11, from pH7 to pH11, from pH8 to pH11, from pH9 to pH11, or from pH10to pH11.5.

The compositions herein may have cleaning activity over a wide range oftemperatures, e.g., from 10° C. or lower to 90° C. Preferably thetemperature will be below 50° C. or 40° C. or even 30° C. In certainaspects, the optimum temperature range for the compositions is from 10°C. to 20° C., from 15° C. to 25° C., from 15° C. to 30° C., from 20° C.to 30° C., from 25° C. to 35° C., from 30° C. to 40° C., from 35° C. to45° C., or from 40° C. to 50° C.

In one aspect a detergent composition may comprise: a source of sulfitesuch as e.g. sodium bisulfite; lipase such as e.g. the lipase of theinvention; linear alkylbenzene sulfonates; C12-16 Pareth-9, propylene;glycol; alcoholethoxy sulfate, water, polyethyleneimine ethoxylate,glycerine; fatty acid salts; PEG-136 polyvinyl acetate; ethylene Diaminedisuccinic salt; monoethanolamine citrate; diethylenetriaminepentaacetate; sodium; disodium distyrylbiphenyl disulfonate; calciumformate; sodium formate; hydrogenated castor oil; dyes;benzisothiazolin; perfume; and optionally one or more enzymes. In oneaspect a detergent composition may comprise: a source of sulfite such ase.g. Potassium Sulfite; lipase such as e.g. the lipase of the invention;MEA-Dodecylbenzenesulfonate; Propylene Glycol; C12-14 Pareth-7; Aqua;MEA-Laureth Sulfate; MEA-Palm Kernelate; PEI Ethoxylate; Glycerin; MEACitrate; PARFUM; Dodecylbenzene Sulfonic Acid; Magnesium Chloride;Disodium Distyrylbiphenyl Disulfonate; PEG/PPG-10/2 Propylheptyl Ether;Hydrogenated Castor Oil; Ethanolamine; Polystyrene; Sodium Formate;Sorbitol; Tripropylene Glycol; 2-Propenoic acid, polymer withethenylbenzene; Butylphenyl Methylpropional; Sulfuric Acid; andoptionally more enzymes.

Form of the Composition

The compositions of the invention described herein may advantageously beemployed for example, in laundry applications, hard surface cleaning,dishwashing applications, as well as cosmetic applications such asdentures, teeth, hair and skin. The compositions of the invention are inparticular solid or liquid cleaning and/or treatment compositions. Inone aspect, the invention relates to a composition, wherein the form ofthe composition is selected from the group consisting of: a regular,compact or concentrated liquid; a gel; a paste; a soap bar; a regular ora compacted powder; a granulated solid; a homogenous or a multilayertablet with two or more layers (same or different phases); a pouchhaving one or more compartments; a single or a multi-compartment unitdose form; or any combination thereof.

The form of the composition may separate the components physically fromeach other in compartments such as e.g. water dissolvable pouches or indifferent layers of tablets. Thereby negative storage interactionbetween components can be avoided. Different dissolution profiles ofeach of the compartments can also give rise to delayed dissolution ofselected components in the wash solution.

Pouches can be configured as single or multicompartments. It can be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume can be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmscan also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches cancomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components can be different incomposition than compartments containing solids (US2009/0011970 A1).

Processes of Making the Compositions

The compositions of the present invention can be formulated into anysuitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in Applicants' examples andin U.S. Pat. No. 4,990,280; US20030087791A1; US20030087790A1;US20050003983A1; US20040048764A1; U.S. Pat. Nos. 4,762,636; 6,291,412;US20050227891A1; EP1070115A2; U.S. Pat. Nos. 5,879,584; 5,691,297;5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; 5,486,303 all ofwhich are incorporated herein by reference. The compositions of theinvention or prepared according to the invention comprise cleaningand/or treatment composition including, but not limited to, compositionsfor treating fabrics, hard surfaces and any other surfaces in the areaof fabric and home care, including: air care including air freshenersand scent delivery systems, car care, dishwashing, fabric conditioning(including softening and/or freshening), laundry detergency, laundry andrinse additive and/or care, hard surface cleaning and/or treatmentincluding floor and toilet bowl cleaners, granular or powder-formall-purpose or “heavy-duty” washing agents, especially cleaningdetergents; liquid, gel or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid types; liquid fine-fabricdetergents; hand dishwashing agents or light duty dishwashing agents,especially those of the high-foaming type; machine dishwashing agents,including the various tablet, granular, liquid and rinse-aid types forhousehold and institutional use: car or carpet shampoos, bathroomcleaners including toilet bowl cleaners; as well as cleaning auxiliariessuch as bleach additives and “stain-stick” or pre-treat types,substrate-laden compositions such as dryer added sheets. Preferred arecompositions and methods for cleaning and/or treating textiles and/orhard surfaces, most preferably textiles. The compositions are preferablycompositions used in a pre-treatment step or main wash step of a washingprocess, most preferably for use in textile washing step.

As used herein, the term “fabric and/or hard surface cleaning and/ortreatment composition” is a subset of cleaning and treatmentcompositions that includes, unless otherwise indicated, granular orpowder-form all-purpose or “heavy-duty” washing agents, especiallycleaning detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, car or carpet shampoos, bathroom cleaners includingtoilet bowl cleaners; fabric conditioning compositions includingsoftening and/or freshening that may be in liquid, solid and/or dryersheet form; as well as cleaning auxiliaries such as bleach additives and“stain-stick” or pre-treat types, substrate-laden compositions such asdryer added sheets. All of such compositions which are applicable may bein standard, concentrated or even highly concentrated form even to theextent that such compositions may in certain aspect be non-aqueous.

Lipase Variants of the Invention

The present invention also relates to lipase variants of a parentlipase, wherein said variant has lipase activity, has at least 60% butless than 100% sequence identity to SEQ ID NO: 2; or any fragmentthereof with lipase activity, and comprises one or more (e.g. several)substitutions at positions corresponding to residues 15, 23, 35, 37, 39,40, 42, 101, 105, 106, 184, 267 of a parent lipase, in particular of SEQID NO: 2.

In a preferred embodiment lipase variant has one or more substitutionscorresponding to the following substitutions: Q15D,E; G23D,E; T35D,E;T37D,E; N39D,E; A40,DE; P42D,E; N101D,E; S105D,E; G106D,E; F184D,E orT267D,E of SEQ ID NO: 2.

In an embodiment, the variant of the parent lipase comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,or 42 substitutions.

In one aspect, the variant has sequence identity of at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, to the amino acid sequence of the parent lipase.

In one aspect, the variant has at least 60%, e.g., at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, suchas at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100%, sequence identity to the polypeptide of SEQ ID NO: 2; SEQ IDNO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12,or a fragement thereof with lipase activity.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-40, 1-30, 1-20, 1-10, 1-5, such as 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40substitutions.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 15 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 15 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 23 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 23 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 35 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 35 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 37 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 37 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 39 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 239 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 40 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 40 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 42 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 42 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 101 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 101 is substituted with E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 105 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 105 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 106 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 106 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 184 of the parent lipase. In one aspect theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 184 is substituted with E or D.

In one aspect, the variant comprises a substitution at a positioncorresponding to position 267 of the parent lipase. In one aspect, theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;OR SEQ ID NO: 10; OR SEQ ID NO: 12. In one aspect, the amino acid at aposition corresponding to position 267 of SEQ ID NO: 2 is substitutedwith E or D.

In one aspect, the variant of a parent lipase comprises a substitutionat two positions corresponding to any of positions: Q15D,E; G23D,E;T35D,E; T37D,E; N39D,E; A40D,E; P42D,E; N101D,E; S105D,E; G106D,E;F184D,E or T267D,E of the parent lipase, in particular of SEQ ID NO: 2.In one aspect the parent lipase has the amino acid sequence of SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; orSEQ ID NO: 12, or a fragment thereof with lipase activity.

In one aspect, the variant of a parent lipase comprises or consists oftwo substitutions at positions corresponding to: Q15D+G23D, Q15D+G23E,Q15D+T35D, Q15D+T35E, Q15D+T37D, Q15D+T37E, Q15D+N39D, Q15D+N39E,Q15D+A40D, Q15D+A40E, Q15D+P42D, Q15D+P42E, Q15D+N101D, Q15D+N101E,Q15D+S105D, Q15D+S105E, Q15D+G106D, Q15D+G106E, Q15D+F184D, Q15D+F184E,Q15D+T267D, Q15D+T267E, Q15E+G23D, Q15E+G23E, Q15E+T35D, Q15E+T35E,Q15E+T37D, Q15E+T37E, Q15E+N39D, Q15E+N39E, Q15E+A40D, Q15E+A40E,Q15E+P42D, Q15E+P42E, Q15E+N101D, Q15E+N101E, Q15E+S105D, Q15E+S105E,Q15E+G106D, Q15E+G106E, Q15E+F184D, Q15E+F184E, Q15E+T267D, Q15E+T267E,G23D+T35D, G23D+T35E, G23D+T37D, G23D+T37E, G23D+N39D, G23D+N39E,G23D+A40D, G23D+A40E, G23D+P42D, G23D+P42E, G23D+N101D, G23D+N101E,G23D+S105D, G23D+S105E, G23D+G106D, G23D+G106E, G23D+F184D, G23D+F184E,G23D+T267D, G23D+T267E, G23E+T35D, G23E+T35E, G23E+T37D, G23E+T37E,G23E+N39D, G23E+N39E, G23E+A40D, G23E+A40E, G23E+P42D, G23E+P42E,G23E+N101D, G23E+N101E, G23E+S105D, G23E+S105E, G23E+G106D, G23E+G106E,G23E+F184D, G23E+F184E, G23E+T267D, G23E+T267E, T35D+T37D, T35D+T37E,T35D+N39D, T35D+N39E, T35D+A40D, T35D+A40E, T35D+P42D, T35D+P42E,T35D+N101D, T35D+N101E, T35D+S105D, T35D+S105E, T35D+G106D, T35D+G106E,T35D+F184D, T35D+F184E, T35D+T267D, T35D+T267E, T35E+T37D, T35E+T37E,T35E+N39D, T35E+N39E, T35E+A40D, T35E+A40E, T35E+P42D, T35E+P42E,T35E+N101D, T35E+N101E, T35E+S105D, T35E+S105E, T35E+G106D, T35E+G106E,T35E+F184D, T35E+F184E, T35E+T267D, T35E+T267E, T37D+N39D, T37D+N39E,T37D+A40D, T37D+A40E, T37D+P42D, T37D+P42E, T37D+N101D, T37D+N101E,T37D+S105D, T37D+S105E, T37D+G106D, T37D+G106E, T37D+F184D, T37D+F184E,T37D+T267D, T37D+T267E, T37E+N39D, T37E+N39E, T37E+A40D, T37E+A40E,T37E+P42D, T37E+P42E, T37E+N101D, T37E+N101E, T37E+S105D, T37E+S105E,T37E+G106D, T37E+G106E, T37E+F184D, T37E+F184E, T37E+T267D, T37E+T267E,N39D+A40D, N39D+A40E, N39D+P42D, N39D+P42E, N39D+N101D, N39D+N101E,N39D+S105D, N39D+S105E, N39D+G106D, N39D+G106E, N39D+F184D, N39D+F184E,N39D+T267D, N39D+T267E, N39E+A40D, N39E+A40E, N39E+P42D, N39E+P42E,N39E+N101D, N39E+N101E, N39E+S105D, N39E+S105E, N39E+G106D, N39E+G106E,N39E+F184D, N39E+F184E, N39E+T267D,N39E+T267E, A40D+P42D, A40D+P42E,A40D+N101D, A40D+N101E, A40D+S105D, A40D+S105E, A40D+G106D, A40D+G106E,A40D+F184D, A40D+F184E, A40D+T267D, A40D+T267E, A40E+P42D, A40E+P42E,A40E+N101D, A40E+N101E, A40E+S105D, A40E+S105E, A40E+G106D, A40E+G106E,A40E+F184D, A40E+F184E, A40E+T267D, A40E+T267E, P42D+N101D, P42D+N101E,P42D+S105D, P42D+S105E, P42D+G106D, P42D+G106E, P42D+F184D, P42D+F184E,P42D+T267D, P42D+T267E, P42E+N101D, P42E+N101E, P42E+S105D, P42E+S105E,P42E+G106D, P42E+G106E, P42E+F184D, P42E+F184E, P42E+T267D,P42E+T267E,N101D+S105D,N101D+S105E,N101D+G106D,N101D+G106E,N101D+F184D,N101D+F184E, N101D+T267D, N101D+T267E, N101E+S105D, N101E+S105E,N101E+G106D, N101E+G106E, N101E+F184D, N101E+F184E, N101E+T267D,N101E+T267E, S105D+S105E, S105D+G106D, S105D+G106E, S105D+F184D,S105D+F184E, S105D+T267D, S105D+T267E, S105E+G106D, S105E+G106E,S105E+F184D, S105E+F184E, S105E+T267D, S105E+T267E, G106D+F184D,G106D+F184E, G106D+T267D, G106D+T267E, G106E+F184D, G106E+F184E,G106E+T267D, G106E+T267E, F184D+T267D, F184D+T267E, F184E+T267D, andF184E+T267E.

In one aspect, the substitutions of the variant of a parent lipasefurther comprises a set of substitutions (using SEQ ID NO: 2 fornumbering) at positions corresponding to:

F51V E96V K98E E210K L259F; F51V S85K K98E; F51V K98E E210V; P250V; F51VE87T K98E E210K I252W L259F; F51V L93K K98E; S83T I86P G91A N92D E96LL97P K98Q; F51V N92K K98E; A40E F51V K98E; F51V E87V K98E; Q15R F51VK98E; F51V E87L K98E; F51V K98E L264P; A40Q F51V K98E; F51V K98E L264W;E210R; F51V E56K L69R K98E I196V; Q4K F51V K98E; F51V G91T K98E; A38LF51V K98E; V2K F51V E56K L69R K98E V230R; F51V G91V K98E; F51V E56K L69RE96T K98E; F51V K98E T189D; F51V K98E R125Q; A40I F51V K98E; F51V K98EK223T; R27D A38G E96D A111D K163G S254D T256P; F51V K98E R133Q; F51VL69C K98E; F51V G91A K98E; F51V G91F K98E; F51V K98E S216P; A40G F51VK98E; F51V K98E N251G; F51V G91Y K98E; F51V K98E V154L; G23S F51V K98E;F51V K98E R108K; E1H F51V K98E; F51V K98E S170T; G23T F51V K98E; F51VK98E T267V; F51V K98E H135Y; F51V K98E L269D; F51V E87K K98E E210QL227G; F51V K98E T267L; F51V K98E R139M; R27D F51V K98E; F51V K98EN162G; E210R; F51V L69R E96V K98E E210K F211W; F51V K98E N251G; R27DF51V K98E; G23H F51V K98E; F51V E96T K98E E210K; E87K; F51V E96D K98EE210K; F51V K98E L264E; F51V L69R E96V K98E E210K F211W; F51V E56K K98EK163P V176L E210K Y220F L227G; F51V E210K; V2K F51V L227G; F51V K98I;F51V K98E E210K F211W L259F; F51V E56K L69R G91A N92D E96L K98Q; F51VE56K L69R K98E I196V; F51V E96T K98E E210K; F51V K98E E210K F211W I252W;F51V E87K K98I; F51V K98E N101D K163P E210K; F51V N101D E210K; F51V E96DK98E E210K; F51V K98E V176L E210K L227G; F51V E96T K98E E210K F211W;F51V E56K L69R E96D K98E; F51V E56K L69R K98E T267L; F51V K98E N101D;F51V E56K L69R K98E A180D; F51V E56K L69R E96D K98E; F51V E56K L69R K98ED165N.

In one aspect, the parent lipase has the amino acid sequence of SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; orSEQ ID NO: 12, or a fragment thereof with lipase activity.

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

In another embodiment the variants further comprise one or more (e.g.,several) alterations such as substitutions corresponding to any ofpositions selected from: 4, 27, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99,111, 150, 163, 210, 216, 225, 227, 231, 233, 249, 254, 255, 256, 263,264, 265, 266, 267, and 269 of the parent lipase, in particular SEQ IDNO: 2. In one aspect the variants further comprise one or more (e.g.,several) alterations such as substitutions corresponding to any ofpositions selected from: 4V, 27R, 38A, 57G, S58A, 60S, 83T, 86V,91A/N/Q, 94K/R, 97M, 99K, 111A, 150G, 163K, 210K/Q, 216P, 225R, L227G,231R, 233R, 249R, 254S, 255A, 256K/TN, 263Q, 264A, 265T, 266D, 267A, and269N of the parent lipase. In one aspect, the parent lipase has theamino acid sequence of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ IDNO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12, or a fragment thereof withlipase activity.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for lipase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

The variants may consist or contain at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% of the number of amino acids of SEQID NO: 2.

In one aspect, the variant has improved stability as compared to theparent lipase. In one aspect, the parent lipase is SEQ ID NO: 2; SEQ IDNO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12.In one aspect, the variant has improved stability in the presence of asource of sulfite as compared to the parent lipase. In one aspect, theparent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;or SEQ ID NO: 10; or SEQ ID NO: 12. The improved stability in thepresence of a source of sulfite may be determined by the “StabilityAssay” as described Example 1.

In one aspect, the variant has improved stability in a detergentcomposition as compared to the parent lipase. In one aspect, the parentlipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQID NO: 10; or SEQ ID NO: 12. In one aspect, the variant has improvedstability in a detergent composition comprising a source of sulfite ascompared to the parent lipase. In one aspect the parent lipase is SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; orSEQ ID NO: 12.

In one aspect, the variant has improved stability under storageconditions as compared to the parent lipase. In one aspect, the parentlipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQID NO: 10; or SEQ ID NO: 12. In one aspect, the variant has improvedstability under storage conditions in the presence of a source sulfiteas compared to the parent lipase. In one aspect, the parent lipase isSEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO:10; or SEQ ID NO: 12.

In one aspect, the variant has improved thermostability as compared tothe parent lipase.

In one aspect, the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ IDNO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect,the variant has improved thermostability in the presence of a sourcesulfite as compared to the parent lipase. In one aspect the parentlipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQID NO: 10; or SEQ ID NO: 12.

Parent Lipase

The parent lipase may be (a) a polypeptide having at least 60% sequenceidentity to the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6;SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12; (b) a polypeptideencoded by a polynucleotide that hybridizes under low stringencyconditions with (i) the polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11,(ii) the full-length complement of (i); or (c) a polypeptide encoded bya polynucleotide having at least 60% sequence identity to thepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

In an aspect, the parent has a sequence identity to the polypeptide ofSEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO:10; or SEQ ID NO: 12 of at least 60%, e.g., at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have lipaseactivity. In one aspect, the amino acid sequence of the parent differsby up to 40 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or40 from the polypeptide of SEQ IDNO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; orSEQ ID NO: 12.

In one aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; orSEQ ID NO: 10; or SEQ ID NO: 12.

In one aspect, the parent is a fragment of the polypeptide of SEQ ID NO:2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12, containing at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% of the number of amino acids of SEQ ID NO: 2; SEQID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO:12.

In one aspect, the parent is an allelic variant of the polypeptide ofSEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO:10; or SEQ ID NO: 12.

In one aspect, the parent is encoded by a polynucleotide that hybridizesunder very low stringency conditions, low stringency conditions, mediumstringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with (i) thepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, (ii) the full-lengthcomplement of (i) (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or a subsequence thereof, as wellas the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ IDNO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12, or a fragment thereof, may beused to design nucleic acid probes to identify and clone DNA encoding aparent from strains of different genera or species according to methodswell known in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, or SEQ ID NO: 11 or a subsequence thereof, the carrier material isused in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, or SEQ ID NO: 11; (ii) the polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ IDNO: 11; (iii) the full-length complement thereof; or (iv) a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the polypeptide coding sequenceof SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,or SEQ ID NO: 11. In one aspect, the nucleic acid probe is at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% of the number ofnucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, or SEQ ID NO: 11. In one aspect, the nucleic acid probe isa polynucleotide that encodes the polypeptide of SEQ ID NO: 2; SEQ IDNO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12;the polypeptide thereof; or a fragment thereof. In one aspect, thenucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

In one aspect, the parent is encoded by a polynucleotide having asequence identity to the polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11of at least 60%, e.g., at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial lipase. For example, the parent may be aGram-positive bacterial polypeptide such as a Bacillus, Clostridium,Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus,Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase, or aGram-negative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma lipase.

In one aspect, the parent is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis lipase.

In one aspect, the parent is a Streptococcus equisimilis, Streptococcuspyogenes, Streptococcus uberis, or Streptococcus equi subsp.Zooepidemicus lipase.

In one aspect, the parent is a Streptomyces achromogenes, Streptomycesavermitilis, Streptomyces coelicolor, Streptomyces griseus, orStreptomyces lividans lipase.

In one aspect, the parent is a Thermobifida alba or Thermobifida fusca(formerly known as Thermomonaspora fusca) lipase.

The parent may be a fungal lipase. For example, the parent may be ayeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipasesuch as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria lipase.

In one aspect, the parent is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis lipase.

In one aspect, the parent is an Acremonium cellulolyticus, Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusfumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Chrysosporium inops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporiumtropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa,Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

In one aspect, the parent is a Thermomyces lanuginosus lipase, e.g., thelipase of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQID NO: 10; or SEQ ID NO: 12.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes.

Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding a parentmay then be obtained by similarly screening a genomic DNA or cDNAlibrary of another microorganism or mixed DNA sample. Once apolynucleotide encoding a parent has been detected with the probe(s),the polynucleotide can be isolated or cloned by utilizing techniquesthat are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a lipasevariant of a parent lipase, wherein the variant has lipase activity,comprises one or more (e.g. several) substitutions at positionscorresponding to position 15, 23, 35, 37, 39, 40, 42, 101, 105, 106,184, 267 of the parent lipase, which parent lipase may have the aminoacid sequence of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;SEQ ID NO: 10; SEQ ID NO: 12; or any fragment thereof with lipaseactivity.

In an embodiment the substitutions are selected from positionscorresponding to: Q15D, E; G23D, E; T35D, E; T37D, E; N39D, E; A40D, E;P42D, E; N101D, E; S105D, E; G106D, E; F184D, E; or T267D, E of SEQ IDNO: 2.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., US2004/0171154; Storici et al., 2001,Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO95/17413; or WO95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryllA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO96/00787), Fusariumvenenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria(WO00/56900), Fusarium venenatum Quinn (WO00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIll, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylllA gene (WO94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to expression vectors, preferably arecombinant expression vectors comprising a polynucleotide encoding avariant of the present invention, a promoter, and transcriptional andtranslational stop signals. The various nucleotide and control sequencesmay be joined together to produce a recombinant expression vector thatmay include one or more convenient restriction sites to allow forinsertion or substitution of the polynucleotide encoding the variant atsuch sites. Alternatively, the polynucleotide may be expressed byinserting the polynucleotide or a nucleic acid construct comprising thepolynucleotide into an appropriate vector for expression. In creatingthe expression vector, the coding sequence is located in the vector sothat the coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMIR1permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M.I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant ofa parent lipase, comprising: (a) cultivating a host cell of the presentinvention under conditions suitable for expression of the variant; and(b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant such as thosedescribed in the examples.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Lipase Particles

The lipase variants of the present invention may be comprised inwater-soluble film as lipase particles. The lipase particles may containone or more additional enzymes, as described below.

Lipase particles are any form of lipase variant in a solid particulateform. That can be as lipase crystals, lipase precipitate, spray orfreeze-dried lipase or any form of granulated lipase, either as a powderor a suspension in liquid. Typically, the particle size, measured asequivalent spherical diameter (volume based average particle size), ofthe lipase particles is below 2 mm, preferably below 1 mm, below 0.5 mm,below 0.25 mm, or below 0.1 mm; and above 0.05 um, preferably above 0.1um, above 0.5 um, above lum, above 5 um or above 10 um. In a preferredaspect, the particle size of the lipase particles is from 0.5 um to 100um.

The lipase particles contain at least 1% w/w lipase protein, preferablyat least 5% w/w lipase protein, at least 10% w/w lipase protein, atleast 20% w/w lipase protein, at least 30% w/w lipase protein, at least40% w/w lipase protein, at least 50% w/w lipase protein, at least 60%w/w lipase protein, at least 70% w/w lipase protein, at least 80% w/wlipase protein, or at least 90% w/w lipase protein.

In a preferred embodiment, the lipase particles are lipase crystals, orthe lipase protein is on a crystalline form. Enzyme crystallization maybe carried out in a number of ways, as known in the art (e.g., asdescribed in WO91/09943 or WO94/22903).

The lipase may be formulated in the lipase particle as known in the artfor solid enzyme formulations, such as formulations for reducing dust,improving stability and/or modifying release rate of the enzyme. Thelipase particle may also be formulated in a matrix or coated with agentssuppressing dissolution of the enzyme particle in the PVOH/film solutionused for preparing the water-soluble film.

The lipase molecules on the surface of the lipase particles may also becross-linked, like CLECs (Cross-Linked Enzyme Crystals) or CLEA(Cross-Linked Enzyme Aggregate).

Water-Soluble Film

Water-soluble films, optional ingredients for use therein, and methodsof making the same are well known in the art. In one class ofembodiments, the water-soluble film includes PVOH. PVOH is a syntheticresin generally prepared by the alcoholysis, usually termed hydrolysisor saponification, of polyvinyl acetate. Fully hydrolyzed PVOH, whereinvirtually all the acetate groups have been converted to alcohol groups,is a strongly hydrogen-bonded, highly crystalline polymer whichdissolves only in hot water—greater than about 140° F. (60° C.). If asufficient number of acetate groups are allowed to remain after thehydrolysis of polyvinyl acetate, the PVOH polymer then being known aspartially hydrolyzed, it is more weakly hydrogen-bonded and lesscrystalline and is soluble in cold water—less than about 50° F. (10°C.). An intermediate cold/hot water-soluble film can include, forexample, intermediate partially-hydrolyzed PVOH (e.g., with degrees ofhydrolysis of about 94% to about 98%), and is readily soluble only inwarm water—e.g., rapid dissolution at temperatures of about 40° C. andgreater. Both fully and partially hydrolyzed PVOH types are commonlyreferred to as PVOH homopolymers although the partially hydrolyzed typeis technically a vinyl alcohol-vinyl acetate copolymer.

The degree of hydrolysis of the PVOH included in the water-soluble filmsmay be about 75% to about 99%. As the degree of hydrolysis is reduced, afilm made from the resin will have reduced mechanical strength butfaster solubility at temperatures below about 20° C. As the degree ofhydrolysis increases, a film made from the resin will tend to bemechanically stronger and the thermoformability will tend to decrease.The degree of hydrolysis of the PVOH can be chosen such that thewater-solubility of the resin is temperature dependent, and thus thesolubility of a film made from the resin, compatibilizing agent, andadditional ingredients is also influenced. In one class of embodimentsthe film is cold water-soluble. A cold water-soluble film, soluble inwater at a temperature of less than 10° C., can include PVOH with adegree of hydrolysis in a range of about 75% to about 90%, or in a rangeof about 80% to about 90%, or in a range of about 85% to about 90%. Inanother class of embodiments the film is hot water-soluble. A hotwater-soluble film, soluble in water at a temperature of at least about60° C., can include PVOH with a degree of hydrolysis of at least about98%.

Other film-forming resins for use in addition to or in an alternative toPVOH can include, but are not limited to, modified polyvinyl alcohols,polyacrylates, water-soluble acrylate copolymers, polyacrylates,polyacryamides, polyvinyl pyrrolidone, pullulan, water-soluble naturalpolymers including, but not limited to, guar gum, xanthan gum,carrageenan, and starch, water-soluble polymer derivatives including,but not limited to, ethoxylated starch and hydroxypropylated starch,poly(sodium acrylamido-2-methylpropane sulfonate),polymonomethylmaleate, copolymers thereof, and combinations of any ofthe foregoing. In one class of embodiments, the film-forming resin is aterpolymer consisting of vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate. Unexpectedly, water-soluble filmsbased on a vinyl alcohol, vinyl acetate, and sodiumacrylamido-2-methylpropanesulfonate terpolymer have demonstrated a highpercent recovery of enzyme.

The water-soluble resin can be included in the water-soluble film in anysuitable amount, for example an amount in a range of about 35 wt % toabout 90 wt %. The preferred weight ratio of the amount of thewater-soluble resin as compared to the combined amount of all enzymes,enzyme stabilizers, and secondary additives can be any suitable ratio,for example a ratio in a range of about 0.5 to about 5, or about 1 to 3,or about 1 to 2.

Water-soluble resins for use in the films described herein (including,but not limited to PVOH resins) can be characterized by any suitableviscosity for the desired film properties, optionally a viscosity in arange of about 5.0 to about 30.0 cP, or about 10.0 cP to about 25 cP.

The viscosity of a PVOH resin is determined by measuring a freshly madesolution using a Brookfield LV type viscometer with UL adapter asdescribed in British Standard EN ISO 15023-2:2006 Annex E BrookfieldTest method. It is international practice to state the viscosity of 4%aqueous polyvinyl alcohol solutions at 20° C. All PVOH viscositiesspecified herein in cP should be understood to refer to the viscosity of4% aqueous polyvinyl alcohol solution at 20° C., unless specifiedotherwise.

It is well known in the art that the viscosity of a PVOH resin iscorrelated with the weight average molecular weight (Mw) of the samePVOH resin, and often the viscosity is used as a proxy for Mw. Thus, theweight average molecular weight of the water-soluble resin optionallycan be in a range of about 35,000 to about 190,000, or about 80,000 toabout 160,000. The molecular weight of the resin need only be sufficientto enable it to be molded by suitable techniques to form a thin plasticfilm.

The water-soluble films may include other optional additive ingredientsincluding, but not limited to, plasticizers, surfactants, defoamers,film formers, antiblocking agents, internal release agents,anti-yellowing agents and other functional ingredients, for example inamounts suitable for their intended purpose.

Water is recognized as a very efficient plasticizer for PVOH and otherpolymers; however, the volatility of water makes its utility limitedsince polymer films need to have at least some resistance (robustness)to a variety of ambient conditions including low and high relativehumidity. Glycerin is much less volatile than water and has been wellestablished as an effective plasticizer for PVOH and other polymers.Glycerin or other such liquid plasticizers by themselves can causesurface “sweating” and greasiness if the level used in the filmformulation is too high. This can lead to problems in a film such asunacceptable feel to the hand of the consumer and even blocking of thefilm on the roll or in stacks of sheets if the sweating is not mitigatedin some manner, such as powdering of the surface. This could becharacterized as over plasticization. However, if too little plasticizeris added to the film the film may lack sufficient ductility andflexibility for many end uses, for example to be converted into a finaluse format such as pouches.

Plasticizers for use in water-soluble films of the present disclosureinclude, but are not limited to, sorbitol, glycerol, diglycerol,propylene glycol, ethylene glycol, diethyleneglycol, triethylene glycol,tetraethyleneglycol, polyethylene glycols up to MW 400, 2 methyl 1, 3propane diol, lactic acid, monoacetin, triacetin, triethyl citrate,1,3-butanediol, trimethylolpropane (TMP), polyether triol, andcombinations thereof. Polyols, as described above, are generally usefulas plasticizers. As less plasticizer is used, the film can become morebrittle, whereas as more plasticizer is used the film can lose tensilestrength. Plasticizers can be included in the water-soluble films in anamount in a range of about 25 phr to about 50 phr, or from about 30 phrto about 45 phr, or from about 32 phr to about 42 phr, for example.

Surfactants for use in water-soluble films are well known in the art.Optionally, surfactants are included to aid in the dispersion of theresin solution upon casting. Suitable surfactants for water-solublefilms of the present disclosure include, but are not limited to, dialkylsulfosuccinates, lactylated fatty acid esters of glycerol and propyleneglycol, lactylic esters of fatty acids, sodium alkyl sulfates,polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkylpolyethylene glycol ethers, lecithin, acetylated fatty acid esters ofglycerol and propylene glycol, sodium lauryl sulfate, acetylated estersof fatty acids, myristyl dimethylamine oxide, trimethyl tallow alkylammonium chloride, quaternary ammonium compounds, salts thereof andcombinations of any of the forgoing. Thus, surfactants can be includedin the water-soluble films in an amount of less than about 2 phr, forexample less than about 1 phr, or less than about 0.5 phr, for example.

One type of secondary component contemplated for use is a defoamer.Defoamers can aid in coalescing of foam bubbles. Suitable defoamers foruse in water-soluble films according to the present disclosure include,but are not limited to, hydrophobic silicas, for example silicon dioxideor fumed silica in fine particle sizes, including Foam Blast® defoamersavailable from Emerald Performance Materials, including Foam Blast® 327,Foam Blast® UVD, Foam Blast® 163, Foam Blast® 269, Foam Blast® 338, FoamBlast® 290, Foam Blast® 332, Foam Blast® 349, Foam Blast® 550 and FoamBlast® 339, which are proprietary, non-mineral oil defoamers. Inembodiments, defoamers can be used in an amount of 0.5 phr, or less, forexample, 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr.Preferably, significant amounts of silicon dioxide will be avoided, inorder to avoid stress whitening.

Processes for making water-soluble articles, including films, includecasting, blow-molding, extrusion and blown extrusion, as known in theart. One contemplated class of embodiments is characterized by thewater-soluble film described herein being formed by casting, forexample, by admixing the ingredients described herein with water tocreate an aqueous mixture, for example a solution with optionallydispersed solids, applying the mixture to a surface, and drying offwater to create a film. Similarly, other compositions can be formed bydrying the mixture while it is confined in a desired shape.

In one contemplated class of embodiments, the water-soluble film isformed by casting a water-soluble mixture wherein the water-solublemixture is prepared according to the steps of:

(a) providing a mixture of water-soluble resin, water, and any optionaladditives excluding plasticizers;

(b) boiling the mixture for 30 minutes;

(c) degassing the mixture in an oven at a temperature of at least 40°C.; optionally in a range of 40° C. to 70° C., e.g., about 65° C.;

(d) adding one or more enzymes, plasticizer, and additional water to themixture at a temperature of 65° C. or less; and

(e) stirring the mixture without vortex until the mixture appearssubstantially uniform in color and consistency; optionally for a timeperiod in a range of 30 minutes to 90 minutes, optionally at least 1hour; and

(f) casting the mixture promptly after the time period of stirring(e.g., within 4 hours, or 2 hours, or 1 hour).

If the enzyme is added to the mixture too early, e.g., with thesecondary additives or resin, the activity of the enzyme may decrease.Without intending to be bound by any particular theory, it is believedthat boiling of the mixture with the enzyme leads to the enzymedenaturing and storing in solution for extended periods of time alsoleads to a reduction in enzyme activity.

In one class of embodiments, high enzyme activity is maintained in thewater-soluble films according to the present disclosure by drying thefilms quickly under moderate to mild conditions. As used herein, dryingquickly refers to a drying time of less than 24 hours, optionally lessthan 12 hours, optionally less than 8 hours, optionally less than 2hours, optionally less than 1 hour, optionally less than 45 minutes,optionally less than 30 minutes, optionally less than 20 minutes,optionally less than 10 minutes, for example in a range of about 6minutes to about 10 minutes, or 8 minutes. As used herein, moderate tomild conditions refer to drying temperatures of less than 170° F. (77°C.), optionally in a range of about 150° F. to about 170° F. (about 66°C. to about 77° C.), e.g., 165° F. (74° C.). As the drying temperatureincreases, the enzymes tend to denature faster, whereas as the dryingtemperature decreases, the drying time increases, thus exposing theenzymes to solution for an extended period of time.

The film is useful for creating a packet to contain a composition, forexample laundry or dishwashing compositions, thereby forming a pouch.The film described herein can also be used to make a packet with two ormore compartments made of the same film or in combination with films ofother polymeric materials. Additional films can, for example, beobtained by casting, blow-molding, extrusion or blown extrusion of thesame or a different polymeric material, as known in the art. In one typeof embodiment, the polymers, copolymers or derivatives thereof suitablefor use as the additional film are selected from polyvinyl alcohols,polyvinyl pyrrolidone, polyalkylene oxides, polyacrylic acid, cellulose,cellulose ethers, cellulose esters, cellulose amides, polyvinylacetates, polycarboxylic acids and salts, polyaminoacids or peptides,polyamides, polyacrylamide, copolymers of maleic/acrylic acids,polysaccharides including starch and gelatin, natural gums such asxanthan, and carrageenans. For example, polymers can be selected frompolyacrylates and water-soluble acrylate copolymers, methylcellulose,carboxymethylcellulose sodium, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin,polymethacrylates, and combinations thereof, or selected from polyvinylalcohols, polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC), and combinations thereof.

The pouches and/or packets may comprise at least one sealed compartment.Thus the pouches may comprise a single compartment or multiplecompartments. The pouches may have regions with and without enzymes. Inembodiments including multiple compartments, each compartment maycontain identical and/or different compositions. In turn, thecompositions may take any suitable form including, but not limited toliquid, solid and combinations thereof (e.g., a solid suspended in aliquid). In some embodiments, the pouches comprise a first, second andthird compartment, each of which respectively contains a differentfirst, second and third composition. In some embodiments, thecompositions may be visually distinct as described in EP 2258820.

The compartments of multi-compartment pouches and/or packets may be ofthe same or different size(s) and/or volume(s). The compartments of thepresent multi-compartment pouches can be separate or conjoined in anysuitable manner. In some embodiments, the second and/or third and/orsubsequent compartments are superimposed on the first compartment. Inone aspect, the third compartment may be superimposed on the secondcompartment, which is in turn superimposed on the first compartment in asandwich configuration. Alternatively, the second and third compartmentsmay be superimposed on the first compartment. However, it is alsoequally envisaged that the first, second and optionally third andsubsequent compartments may be attached to one another in a side by siderelationship. The compartments may be packed in a string, eachcompartment being individually separable by a perforation line. Henceeach compartment may be individually torn-off from the remainder of thestring by the end-user.

In some embodiments, multi-compartment pouches and/or packets includethree compartments consisting of a large first compartment and twosmaller compartments. The second and third smaller compartments aresuperimposed on the first larger compartment. The size and geometry ofthe compartments are chosen such that this arrangement is achievable.The geometry of the compartments may be the same or different. In someembodiments, the second and optionally third compartment each has adifferent geometry and shape as compared to the first compartment. Inthese embodiments, the second and optionally third compartments arearranged in a design on the first compartment. The design may bedecorative, educative, or illustrative, for example to illustrate aconcept or instruction, and/or used to indicate origin of the product.In some embodiments, the first compartment is the largest compartmenthaving two large faces sealed around the perimeter, and the secondcompartment is smaller covering less than about 75%, or less than about50% of the surface area of one face of the first compartment. Inembodiments in which there is a third compartment, the aforementionedstructure may be the same but the second and third compartments coverless than about 60%, or less than about 50%, or less than about 45% ofthe surface area of one face of the first compartment.

The pouches and/or packets may comprise one or more different films. Forexample, in single compartment embodiments, the packet may be made fromone wall that is folded onto itself and sealed at the edges, oralternatively, two walls that are sealed together at the edges. Inmultiple compartment embodiments, the packet may be made from one ormore films such that any given packet compartment may comprise wallsmade from a single film or multiple films having differing compositions.In one aspect, a multi-compartment pouch comprises at least three walls:an outer upper wall; an outer lower wall; and a partitioning wall. Theouter upper wall and the outer lower wall are generally opposing andform the exterior of the pouch. The partitioning wall is interior to thepouch and is secured to the generally opposing outer walls along a sealline. The partitioning wall separates the interior of themulti-compartment pouch into at least a first compartment and a secondcompartment. In one class of embodiments, the partitioning wall may bethe only enzyme containing film thereby minimizing the exposure of theconsumer to the enzymes.

Pouches and packets may be made using any suitable equipment and method.For example, single compartment pouches may be made using vertical formfilling, horizontal form filling, or rotary drum filling techniquescommonly known in the art. Such processes may be either continuous orintermittent. The film may be dampened, and/or heated to increase themalleability thereof. The method may also involve the use of a vacuum todraw the film into a suitable mold. The vacuum drawing the film into themold can be applied for about 0.2 to about 5 seconds, or about 0.3 toabout 3, or about 0.5 to about 1.5 seconds, once the film is on thehorizontal portion of the surface. This vacuum can be such that itprovides an under-pressure in a range of 10 mbar to 1000 mbar, or in arange of 100 mbar to 600 mbar, for example.

The molds, in which packets may be made, can have any shape, length,width and depth, depending on the required dimensions of the pouches.The molds may also vary in size and shape from one to another, ifdesirable. For example, the volume of the final pouches may be about 5mL to about 300 mL, or about 10 mL to 150 mL, or about 20 mL to about100 mL, and that the mold sizes are adjusted accordingly.

In one aspect, the packet includes a first and a second sealedcompartment. The second compartment is in a generally superposedrelationship with the first sealed compartment such that the secondsealed compartment and the first sealed compartment share a partitioningwall interior to the pouch.

In one aspect, the packet including a first and a second compartmentfurther includes a third sealed compartment. The third sealedcompartment is in a generally superposed relationship with the firstsealed compartment such that the third sealed compartment and the firstsealed compartment share a partitioning wall interior to the pouch.

In various aspects, the first composition and the second composition areselected from one of the following combinations: liquid, liquid; liquid,powder; powder, powder; and powder, liquid.

In various aspects, the first, second and third compositions areselected from one of the following combinations: solid, liquid, liquidand liquid, liquid, liquid.

In one aspect, the single compartment or plurality of sealedcompartments contains a composition. The plurality of compartments mayeach contain the same or a different composition. The composition isselected from a liquid, solid or combination thereof.

Heat can be applied to the film in the process commonly known asthermoforming. The heat may be applied using any suitable means. Forexample, the film may be heated directly by passing it under a heatingelement or through hot air, prior to feeding it onto a surface or onceon a surface. Alternatively, it may be heated indirectly, for example byheating the surface or applying a hot item onto the film. The film canbe heated using an infrared light. The film may be heated to atemperature of at least 50° C., for example about 50 to about 150° C.,about 50 to about 120° C., about 60 to about 130° C., about 70 to about120° C., or about 60 to about 90° C.

Alternatively, the film can be wetted by any suitable means, for exampledirectly by spraying a wetting agent (including water, a solution of thefilm composition, a plasticizer for the film composition, or anycombination of the foregoing) onto the film, prior to feeding it ontothe surface or once on the surface, or indirectly by wetting the surfaceor by applying a wet item onto the film.

Once a film has been heated and/or wetted, it may be drawn into anappropriate mold, preferably using a vacuum. The film can bethermoformed with a draw ratio of at least about 1.5, for example, andoptionally up to a draw ratio of 2, for example. The filling of themolded film can be accomplished by utilizing any suitable means. In someembodiments, the most preferred method will depend on the product formand required speed of filling. In some embodiments, the molded film isfilled by in-line filling techniques. The filled, open packets are thenclosed forming the pouches, using a second film, by any suitable method.This may be accomplished while in horizontal position and in continuous,constant motion. The closing may be accomplished by continuously feedinga second film, preferably water-soluble film, over and onto the openpackets and then preferably sealing the first and second film together,typically in the area between the molds and thus between the packets.

Any suitable method of sealing the packet and/or the individualcompartments thereof may be utilized. Non-limiting examples of suchmeans include heat sealing, solvent welding, solvent or wet sealing, andcombinations thereof. The water-soluble packet and/or the individualcompartments thereof can be heat sealed at a temperature of at least200° F. (93° C.), for example in a range of about 220° F. (about 105°C.) to about 290° F. (about 145° C.), or about 230° F. (about 110° C.)to about 280° F. (about 140° C.). Typically, only the area which is toform the seal is treated with heat or solvent. The heat or solvent canbe applied by any method, typically on the closing material, andtypically only on the areas which are to form the seal. If solvent orwet sealing or welding is used, it may be preferred that heat is alsoapplied. Preferred wet or solvent sealing/welding methods includeselectively applying solvent onto the area between the molds, or on theclosing material, by for example, spraying or printing this onto theseareas, and then applying pressure onto these areas, to form the seal.Sealing rolls and belts as described above (optionally also providingheat) can be used, for example.

The formed pouches may then be cut by a cutting device. Cutting can beaccomplished using any known method. It may be preferred that thecutting is also done in continuous manner, and preferably with constantspeed and preferably while in horizontal position. The cutting devicecan, for example, be a sharp item, or a hot item, or a laser, whereby inthe latter cases, the hot item or laser ‘burns’ through the film/sealingarea.

The different compartments of a multi-compartment pouches may be madetogether in a side-by-side style wherein the resulting, cojoined pouchesmay or may not be separated by cutting. Alternatively, the compartmentscan be made separately.

In some embodiments, pouches may be made according to a processincluding the steps of:

a) forming a first compartment (as described above);

b) forming a recess within some or all of the closed compartment formedin step (a), to generate a second molded compartment superposed abovethe first compartment;

c) filling and closing the second compartments by means of a third film;

d) sealing the first, second and third films; and

e) cutting the films to produce a multi-compartment pouch.

The recess formed in step (b) may be achieved by applying a vacuum tothe compartment prepared in step (a).

In some embodiments, second, and/or third compartment(s) can be made ina separate step and then combined with the first compartment asdescribed in EP2088187 or WO2009/152031.

In other embodiments, pouches may be made according to a processincluding the steps of:

a) forming a first compartment, optionally using heat and/or vacuum,using a first film on a first forming machine;

b) filling the first compartment with a first composition;

c) on a second forming machine, deforming a second film, optionallyusing heat and vacuum, to make a second and optionally third moldedcompartment;

d) filling the second and optionally third compartments;

e) sealing the second and optionally third compartment using a thirdfilm;

f) placing the sealed second and optionally third compartments onto thefirst compartment;

g) sealing the first, second and optionally third compartments; and

h) cutting the films to produce a multi-compartment pouch.

The first and second forming machines may be selected based on theirsuitability to perform the above process. In some embodiments, the firstforming machine is preferably a horizontal forming machine, and thesecond forming machine is preferably a rotary drum forming machine,preferably located above the first forming machine.

It should be understood that by the use of appropriate feed stations, itmay be possible to manufacture multi-compartment pouches incorporating anumber of different or distinctive compositions and/or different ordistinctive liquid, gel or paste compositions.

Method of Use

The present invention includes a method for cleaning any surfaceincluding treating a textile or a hard surface or other surfaces in thefield of fabric and/or home care. It is contemplated that cleaning asdescribed may be both in small scale as in e.g. family house hold aswell as in large scale as in e.g. industrial and professional settings.In one aspect of the invention, the method comprises the step ofcontacting the surface to be treated in a pre-treatment step or mainwash step of a washing process, most preferably for use in a textilewashing step or alternatively for use in dishwashing including bothmanual as well as automated/mechanical dishwashing. In one aspect of theinvention the lipase variant and other components are added sequentiallyinto the method for cleaning and/or treating the surface. Alternatively,the lipase variant and other components are added simultaneously.

As used herein, washing includes but is not limited to, scrubbing, andmechanical agitation. Washing may be conducted with a foam compositionas described in WO08/101958 and/or by applying alternating pressure(pressure/vaccum) as an addition or as an alternative to scrubbing andmechanical agitation. Drying of such surfaces or fabrics may beaccomplished by any one of the common means employed either in domesticor industrial settings. The cleaning compositions of the presentinvention are ideally suited for use in laundry as well as dishwashingapplications. Accordingly, the present invention includes a method forcleaning an object including but not limiting to fabric, tableware,cutlery and kitchenware. The method comprises the steps of contactingthe object to be cleaned with a said cleaning composition comprising atleast one aspect of Applicants' cleaning composition, cleaning additiveor mixture thereof. The fabric may comprise most any fabric capable ofbeing laundered in normal consumer or institutional use conditions. Thesolution may have a pH from 8 to 10.5. The compositions may be employedat concentrations from 500 to 15.000 ppm in solution. The watertemperatures typically range from 5° C. to 90° C. The water to fabricratio is typically from 1:1 to 30:1.

In one aspect the invention relates to a method of using a lipasevariant of a parent lipase which parent lipase has the amino acidsequence of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8, SEQID NO: 10; or SEQ ID NO: 12, wherein the variant has lipase activity,comprises a substitution at a position corresponding to position 15, 23,35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2 forproducing a composition.

In one aspect the substitutions are selected from positions Q15D,E;G23D,E; T35D,E; T37D,E; N39D,E; A40D,E; P42D,E; N101 D,E; S105D,E;G106D,E; F184D,E; or T267D,E of SEQ ID NO: 2 of the parent lipase. Inone aspect, the invention relates to use of the composition for cleaningan object.

In one aspect, the invention relates to a method for cleaning a surface,comprising contacting a lipid stain present on the surface to be cleanedwith the cleaning composition. In one aspect, the invention relates to amethod for hydrolyzing a lipid present in a soil and/or a stain on asurface, comprising contacting the soil and/or the stain with thecleaning composition. In one aspect, the invention relates to use of thecomposition in the hydrolysis of a carboxylic acid ester. In one aspect,the invention relates to use of the composition in the hydrolysis,synthesis or interesterification of an ester. In one aspect, theinvention relates to use of the composition for the manufacture of astable formulation.

The present invention is described in below paragraph embodiments:

1. A composition comprising:

(a) a source of sulfite;

(b) a lipase variant of the parent lipase shown as SEQ ID NO: 2 or of aparent lipase having at least 60% identity to SEQ ID NO: 2;

wherein one or more amino acids in the parent lipase within 20 angstrom(A) of a cysteine bridge are replaced with a more negatively chargedamino acid.

2. The composition of paragraph 1, wherein the replaced, preferablysubstituted, amino acid is within 15 angstrom, preferably within 10angstrom, more preferably within 5 angstrom (i.e., from carboxyl groupto sulfur atom) of a cysteine bridge.

3. The composition according paragraph 1 or 2 wherein the source ofsulfite is selected from: sulfites, such as sodium sulfite, potassiumsulfite; calcium sulfite; bisulfite such as potassium bisulfite sodiumbisulfite, calcium bisulfite; metabisulfite such as potassiummetabisulfite, sodium metabisulfite, calcium metabisulfite; or anycombination thereof.4. The composition according to any one of paragraphs 1-3 wherein theamount of sulfite (as SO₃ ²⁻) or metabisulfite (as S₂O₅ ²⁻) is from 0.1wt % to 3 wt %; from 0.2 wt % to 2 wt %; from 0.5 wt % to 2 wt %.5. The composition according to any one of paragraphs 1-4 wherein thelipase variant is a variant of the parent lipase shown as SEQ ID NO: 2or of a lipase having at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, but less than 100%sequence identity to the parent lipase, in particular any of the parentlipases shown as SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8;or SEQ ID NO: 10; or SEQ ID NO: 12, or a fragment thereof with lipaseactivity.6. The composition of any of paragraphs 1-4, wherein the lipase variantfurther comprises one or more (e.g. several) substitutions at positionscorresponding to amino acid residues 15, 23, 35, 37, 39, 40, 42, 101,105, 106, 184, 267 of SEQ ID NO: 2.7. The composition according to any of paragraphs 1-6, wherein the aminoacid in the parent lipase within 20 angstrom (A) of a cysteine bridge isreplaced with a negatively charged amino acid selected from D or E.8. The composition according to any of paragraphs 1-7, wherein thelipase variant comprises substitutions corresponding to Q15D, E; G23D,E; T35D, E; T37D, E; N39D, E; A40D, E; P42D, E; N101D, E; S105D, E;G106D, E; F184D, E or T267D, E of SEQ ID NO: 2.9. The composition according to any one of paragraphs 1-8, wherein theparent lipase has at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ IDNO: 12.10. The composition according to any one of paragraphs 1-9, wherein thecysteine bridge is on the surface of the parent lipase.11. The composition according to any one of paragraphs 1-10, wherein thecysteine bridge is or corresponds to one or more of the cysteine bridgesin positions:C22-C268,C36-C41, andC104-C107of SEQ ID NO: 2.12. The composition of any of paragraphs 1-11 wherein the lipase varianthas improved stability in the presence of a source of sulfite ascompared to the parent lipase, in particular SEQ ID NO: 2.13. The composition according to any one of paragraphs 1-12, wherein thevariant of a parent lipase is selected from the group consisting of:

-   -   (a) a polypeptide encoded by a polynucleotide that hybridizes        under low stringency conditions, medium stringency conditions,        medium-high stringency conditions, high stringency conditions,        or very high stringency conditions with (i) the polypeptide        coding sequence of SEQ ID NO: 1, or (ii) the full-length        complement of (i);    -   (b) a polypeptide encoded by a polynucleotide having at least        60%, at least 65%, at least 70%, at least 75%, at least 80%, at        least 85%, at least 90%, at least 95%, at least 96%, at least        97%, at least 98%, at least 99%, but less that 100% sequence        identity to the polypeptide coding sequence of SEQ ID NO: 1; and    -   (c) a fragment of the polypeptide of (a) or (b), which has        lipase activity.        14. The composition according to any one of paragraphs 1-13,        wherein the variant of a parent lipase comprises 1,2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12 substitutions.        15. The composition according to any one of paragraph 1-14,        wherein the composition further comprising a surfactant.        16. Use of the composition according to any one of paragraphs        1-15 for cleaning a surface including a fabric, textile or hard        surface.        17. A lipase variant of a parent lipase, wherein said variant        has lipase activity, has at least 60% but less than 100%        sequence identity to SEQ ID NO: 2; or any fragment thereof with        lipase activity, and comprises one or more (e.g. several)        substitutions at positions corresponding to residues 15, 23, 35,        37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2.        18. The variant according to paragraph 17, wherein the variant        has one or more substitutions corresponding to the following        substitutions: Q15D, E; G23D, E; T35D, E; T37D, E; N39D, E;        A40D, E; P42D, E; N101D, E; S105D, E; G106D, E; F184D, E or        T267D, E of SEQ ID NO: 2.        19. A polynucleotide encoding the variant according to any one        of paragraph 17 or 18.        20. A nucleic acid construct comprising the polynucleotide        according to paragraph 19.        21. An expression vector comprising the polynucleotide according        to paragraph 19.        22. A host cell comprising the polynucleotide according to        paragraph 19.        23. A method of producing a lipase variant, comprising: (a)        cultivating the host cell according to paragraph 22 under        conditions suitable for expression of the variant; and (b)        recovering the variant.        24. A method for obtaining the lipase variant according to any        one of paragraph 17 or 18, comprising introducing into the        parent lipase shown as SEQ ID NO: 2 or a parent lipase having at        least 60% sequence identity thereto within 20 angstrom (A) of a        cysteine bridge, a more negatively charged amino acid.        25. The method according to paragraph 24, wherein introduced,        preferably substituted, amino acid is within 15 angstrom,        preferably within 10 angstrom, more preferably within 5 angstrom        (i.e., from carboxyl group to sulfur atom) of a cysteine bridge.        26. The method of paragraph 24 or 25, wherein the substitution        to a more negatively charged amino acid is done at a position        corresponding to positions: 15, 23, 35, 37, 39, 40, 42, 101,        105, 106, 184, 267 of SEQ ID NO: 2.        27. The method of any of paragraphs 24-26, wherein the        negatively charged amino acid is D or E.        28. The method of any of paragraphs 24-27, wherein one or more        of the substitutions corresponding to the following        substitutions are introduced into the parent lipase: Q15D,E;        G23D,E; T35D,E; T37D,E; N39D,E; A40D,E; P42D,E; N101D,E;        S105D,E; G106D,E; F184D,E; or T267D,E of SEQ ID NO: 2.        Materials & Methods        p-Nitrophenyl (pNP) Assay:

The hydrolytic activity of a lipase may be determined by a kinetic assayusing p-nitrophenyl acyl esters as substrate.

A 100 mM stock solution in DMSO of the substrates: p-Nitrophenylbutyrate (C₄), p-Nitrophenyl caproate (C6), p-Nitrophenyl caprate (C10),p-Nitrophenyl laurate (C12) and p-Nitrophenyl palmitate (C16) (all fromSigma-Aldrich Danmark A/S, Kirkebjerg Alle 84, 2605 Brondby; Cat.no.:C4:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) may bediluted to a final concentration of 1 mM 25 into assay buffer (50 mMTris; pH 7.7; 0.4% TritonX-100).

The lipase of the invention, the parent lipase and appropriate controlse.g. Buffer (negative), Lipolase™ & Lipex™ (positive) in 50 mM Hepes; pH8.0; 10 ppm TritonX-100; +/−20 mM CaCl₂ may be added to the substratesolution in the following final concentrations: 0.01 mg/mL; 5×10−3mg/mL; 2.5×10−4 mg/mL; and 1.25×10−4 mg/ml in 96-well NUNC plates (Cat.No:260836, Kamstrupvej 90, DK-4000, Roskilde). Release of p-nitrophenolby hydrolysis of p-nitrophenyl acyl may be monitored at 405 nm for 5minutes in 10 second intervals on a Spectra max 190 (Molecular DevicesGmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY). Thehydrolytic activity towards one or more substrates of a variant may becompared to that of the parent lipase.

EXAMPLES Example 1

Stability of Lipase Variants in Detergent Compositions with and withoutSulfite

Lipase variants of SEQ ID NO: 2 were constructed and expressed inAspergillus oryzae, as described in WO 2016/050661 (incorporated byreference).

Growth of Variants:

Transformants expressing lipase variants were inoculated in 96 well MTPwith 200 μl YP+2% maltose. They were grown 120 hours at 34° C. (noshaking).

The variants were screened the following way:

1 g of sodium sulfite was added to 100 g of detergent composition (TIDE™Original Scent HE Turbo Clean Liquid Laundry Detergent from P&G boughtin the USA in March 2017) and stirred for minimum 10 Minutes.™

An aliquot of 90 μL detergent with sulfite was added into a 96 wellplate (with room for 500 μL). An aliquot 90 μL detergent without sulfitewas added into a 96 well plate (with room for 500 μL). Magnets wereadded to the plate.

10 μL of medium from the growth plate was added to each of the detergentplates.

The plates were sealed with alu-seal and shaked on the magnetic platestirrer for at least 10 min. The plates were incubated in a plastic boxat 49° C. in heating cabinet for 19 hours.

Stability Assay:

Dilution buffer: 0.1 M Tris-HCl, 9 mM CaCl₂, 0.0225% Brij-30, pH 8.0

Assay-buffer: AOS-substrate buffer: 100 mM Tris, 6.5 mM Deoxycholate,1.4 g/L AOS buffer pH 8.0

Substrate: 100 mL AOS buffer, 0.5 mM pNP-palimitate, 1 mM CaCl2

230 μL dilution buffer was added to the incubated plates. Detergentplates were stirred for 10 min. 5 μL from detergent plate was added to anew plate containing 295 μL dilution buffer and shaken for 10 min.

101 μL was taken to a 384 well plate from the two former plates (withand without sulfite). 40 μL of substrate solution was added to theplate, and run for 30 min in an Elisa reader.

The following variants of SEQ ID NO: 2 had improved stability (i.e.,increased residual activity) in a detergent composition with sulfite:T35D, N39E, P42D, N101D, S105E, F184D.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. A composition comprising: (a) a source ofsulfite; and (b) a lipase variant which (i) comprises a substitution atone or more positions corresponding to positions 35, 39, 42, and 105, ofSEQ ID NO: 2 with a more negatively charged amino acid; (ii) has atleast 80% but less than 100% sequence identity to SEQ ID NO: 2; and(iii) has lipase activity.
 2. The composition of claim 1, wherein thelipase variant has at least 85% sequence identity to SEQ ID NO:
 2. 3.The composition of claim 1, wherein the lipase variant has at least 90%sequence identity to SEQ ID NO:
 2. 4. The composition of claim 1,wherein the lipase variant has at least 95% sequence identity to SEQ IDNO:
 2. 5. The composition of claim 1, wherein the lipase variantcomprises one or more of the following substitutions: T35D, E; N39D, E;P42D, E; or S105D, E of SEQ ID NO:
 2. 6. The composition of claim 1,wherein the lipase variant comprises T35D.
 7. The composition of claim1, wherein the lipase variant comprises N39E.
 8. The composition ofclaim 1, wherein the lipase variant comprises P42D.
 9. The compositionof claim 1, wherein the lipase variant comprises S105E.
 10. Thecomposition of claim 1, wherein the lipase variant further comprises asubstitution at one or more positions corresponding to positions 4, 27,38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225,227, 231, 233, 249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 ofSEQ ID NO:
 2. 11. The composition of claim 10, wherein the substitutionis selected from the group consisting of 4V, 27R, 38A, 57G, S58A, 60S,83T, 86V, 91A/N/Q, 94K/R, 97M, 99K, 111A, 150G, 163K, 210K/Q, 216P,225R, L227G, 231R, 233R, 249R, 254S, 255A, 256K/T/V, 263Q, 264A, 265T,266D, 267A, and 269N.
 12. The composition of claim 1, wherein the lipasevariant has one or more cysteine bridges at positions corresponding topositions C22-C268, C36-C41, and C104-C107 of SEQ ID NO:
 2. 13. Thecomposition of claim 1, wherein the lipase variant has improvedstability in the presence of the source of sulfite as compared to thelipase of SEQ ID NO:
 2. 14. The composition of claim 1, wherein thesource of sulfite is sodium sulfite, potassium sulfite; calcium sulfite;potassium bisulfite sodium bisulfite, calcium bisulfite; potassiummetabisulfite, sodium metabisulfite, calcium metabisulfite; or anycombination thereof.
 15. The composition of claim 14, wherein the amountof sulfite (as SO₃ ²⁻) or metabisulfite (as S₂O₅ ²⁻) is from 0.1 wt. %to 3 wt. %.
 16. The composition of claim 1, further comprising asurfactant.
 17. A method of cleaning a surface, comprising treating thesurface with a composition of claim
 1. 18. The method of claim 17,wherein the surface is a textile or hard surface.